WO2020232512A1

WO2020232512A1 - Microglial cells and methods of use thereof

Info

Publication number: WO2020232512A1
Application number: PCT/AU2020/050513
Authority: WO
Inventors: Anthony Robert White; Hazel Qian Yin QUEK
Original assignee: The Council Of The Queensland Institute Of Medical Research
Priority date: 2019-05-22
Filing date: 2020-05-22
Publication date: 2020-11-26

Abstract

Disclosed are microglial cells. More particularly, the invention relates to microglial cells generated from blood-derived cells and their use in methods of diagnosing, treating and/or determining the responsiveness to treatment and/or prognosis of neurodegenerative diseases, disorders and conditions.

Description

TITLE

MICROGLIAL CELLS AND METHODS OF USE THEREOF

RELATED APPLICATIONS

This application claims priority to Australian Provisional Application No. 2019 entitled“MICROGLIAL CELLS AND METHODS OF USE THEREOF”, filed on 22 May 2019, the entire content of which is hereby incorporated herein by reference in its entirety.

FIELD

THIS INVENTION relates to microglial cells. More particularly, this invention relates to microglial cells generated from blood-derived cells and their use in methods of diagnosing, treating and/or determining the responsiveness to treatment and/or prognosis of neurodegenerative diseases, disorders and conditions, such as Alzheimer’s disease.

BACKGROUND

The global impact of Alzheimer’s disease is growing rapidly. It is an intractable neurological disorder characterized by accumulation of extracellular amyloid plaques, intracellular neurofibrillary tangles of hyperphosphorylated microtubule tau protein, neuroinflammation, and neuronal loss in the brain. Alzheimer’s is the most common form of dementia in the elderly and is one of the leading contributors to global disease burden with ~50 million dementia patients worldwide, however, there are no effective disease modifying treatments. Over 400 clinical trials in Alzheimer’s patients have had little success at improving cognition or quality of life with the last drug for Alzheimer’s disease approved in 2003. Although amyloid reduction trials in prodromal Alzheimer’s patients may yield prophylactic benefits, the outcomes will not be known for many years. As a consequence, there remains a critical need for improved understanding of Alzheimer’s disease mechanistic pathways and new therapeutic options.

Neuroinflammation is a major contributor to Alzheimer’s disease pathology, however classical anti-inflammatory drugs have been largely ineffective in treating the disease. The immune system is an important mediator of pathogenesis in Alzheimer’s disease, with chronically increased levels of toxic cytokines that inhibit synapse and memory function (i.e., Clq, TNF-a, IFN-g and IL- 1 b ) [ 1 ] . This inflammatory response is largely mediated by microglia, the resident immune cells of the central nervous system. Phagocytic removal of amyloid peptide and cell debris from dying cells is a major function of microglia and inefficient clearance of amyloid has been identified as a major pathogenic change in Alzheimer’s disease brain. [2], [3] Additional critical microglial functions include regulation of synaptic maturation, affecting learning and memory. This failure may be due to the neuroinflammatory response being more complex than a simple over-activation that can be targeted by‘anti-inflammatories’, and clinical trials target a broad cross-section of Alzheimer’s disease patients regardless of risk gene status, evidence of neuroinflammation, and stage of disease progression. [2]-[5] However, clinical trials using classical anti-inflammatory drugs such as Naproxen and minocycline have shown little benefit in Alzheimer’s disease patients. [6] The role of microglia is also thought to change throughout the disease course. [6] In response to these issues, there is an urgent need to understand the complex role of microglia and identify drug targets that are more specific to regulating key aspects of microglia behaviour in Alzheimer’s disease. [6]

In addition to Alzheimer’s, there is also an urgent need for new therapeutic approaches to treat Amyotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disease characterized by the degeneration of both upper and lower motor neurons leading to motor and extra-motor symptoms. The disease normally results in death after 2-3 years due to respiratory failure. ALS affects 2-3 people per 100,000 and has a high degree of care burden on family and public health systems. There are limited treatments that may extend life by 2-3 months. There has been a‘re-awakening’ to the critical role of microglia in mediating pathological neuroinflammation in ALS. However, our ability to translate this knowledge into clinically relevant advances has been largely unsuccessful. This failure is due, in part, to clinical trials targeting a broad cross-section of ALS patients without taking into account risk gene status, evidence of inflammation, stage of disease, and additional factors. To greatly improve the identification of drugs that can modify neuroinflammation in specific ALS patients we need an assay that can identify inflammatory drug efficacy on a patient-by-patient basis.

To this end, current neuroinflammatory cell models have major deficiencies when applied to therapeutic drug screening for neurodegenerative diseases such as Alzheimer’s disease and ALS. Research into microglia function in Alzheimer’s disease, ALS and other neurodegenerative diseases is greatly impeded by lack of ready access to human microglia. Most studies are conducted on rodent microglia, however microglia from humans and rodents behave very differently with critical variances in gene expression profiles between animal disease models and patients. [7], [8] Recent advances in induced pluripotent stem cell (iPSC) technologies have now allowed generation of human microglia and this can be applied to neurodegenerative disease patients. [9], [10] However, the process is time-consuming (months), costly (up to AUD$ 10,000/cell line), and remains problematic due to somatic variation and genomic instability. [10], [11] Due to costs, time-frame and variability, iPSC-derived microglia are not yet suitable for real time patient-based drug screening on a clinically-relevant scale. Accordingly, improved assays in this regard are required.

SUMMARY

The present invention broadly relates to producing microglial cells from blood- derived cells and their use in methods of diagnosing, treating and/or determining the responsiveness to treatment and/or prognosis of neurodegenerative diseases, disorders and conditions. In a particular form, the neurodegenerative disease, disorder or condition is Alzheimer’s disease, Parkinson’s disease or ALS.

In a first aspect, the invention provides a method of producing a microglial cell from a monocyte of a subject, the method including the step of culturing the monocyte with one or a plurality of neural stem cells to thereby produce the microglial cell.

In one embodiment, the method of the present aspect further includes the initial step of isolating the monocyte from the subject. In some embodiments, the monocyte is obtained from peripheral mononuclear cell sample.

In certain embodiments, the current method further includes the subsequent step of collecting or harvesting the microglial cell.

In some embodiments, the neural stem cells are or comprise immortalized or stable neural stem cells and/or embryonic stem cell-derived neural stem cells.

In other embodiments, the blood-derived cell and the neural stem cells are cultured, at least in part, in a 3 -dimensional co-culture system.

Suitably, the neural stem cells are or comprise a brain organoid.

In particular embodiments, the monocyte is cultured with the one or plurality of neural stem cells substantially in the absence of interleukin-34 (IL-34) and granulocyte- macrophage colony stimulating factor (GM-CSF).

In a second aspect, the invention provides a method of diagnosing a neurodegenerative disease, disorder or condition in a subject, including the step of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from monocytes isolated from the subject, wherein the level of phagocytosis, cytokine production and/or migration is altered or modulated in the microglial cells.

In a third aspect, the invention provides a method of determining a prognosis for a neurodegenerative disease, disorder or condition in a subject, said method including the step of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from monocytes isolated from the subject, wherein the level of phagocytosis, cytokine production and/or migration indicates or correlates with a less or more favourable prognosis for the neurodegenerative disease, disorder or condition.

In one embodiment, if the level of phagocytosis, cytokine production and/or migration is altered or modulated in the microglial cells, the prognosis may be negative or positive.

Suitably, the prognosis is used, at least in part, to develop a treatment strategy for the subject.

In other embodiments, the prognosis is used, at least in part, to determine disease progression in the subject.

In one embodiment, the present method further includes the step of determining suitability of the subject for treatment with a therapeutic agent based, at least in part, on the prognosis.

Referring to the second and third aspects, the method further includes the step determining a disease stage and/or grade for the neurodegenerative disease, disorder or condition based on, at least in part, the level of phagocytosis, cytokine production and/or migration.

In a fourth aspect, the invention provides a method of predicting the responsiveness of a neurodegenerative disease, disorder or condition to a therapeutic agent in a subject, said method including the step of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from blood-derived cells isolated from the subject, wherein the level of phagocytosis, cytokine production and/or migration indicates or correlates with relatively increased or decreased responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent.

In one embodiment, the present method further includes the step of administering to the subject a therapeutically effective amount of the therapeutic agent when the level of phagocytosis, cytokine production and/or migration indicates or correlates with relatively increased responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent.

In a fifth aspect, the invention provides a method of treating a neurodegenerative disease, disorder or condition in a subject, the method including the step of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from blood-derived cells isolated from the subject and based on the determination made, initiating, continuing, modifying or discontinuing administration of a therapeutic agent to the subject.

In a sixth aspect, the invention provides a method of stratifying a subject having a neurodegenerative disease, disorder or condition for a clinical trial of a therapeutic agent including the steps of:

determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from blood-derived cells isolated from the subject; and,

stratifying the subject for the clinical trial based on the results of the determining step.

Referring to the fourth, fifth and sixth aspects, the method suitably further includes the step of contacting the microglial cells with the therapeutic agent.

Suitably, the method of the second, third, fourth, fifth and sixth aspects further includes the step of contacting the microglial cells with a stimulant. Preferably, the stimulant is an immune stimulant, such as a Damage-associated molecular pattern (DAMP). In particular embodiments, the stimulant is selected from the group consisting of a beta amyloid peptide, a-synuclein a tau protein, TDP-43, SOD1, a lipopoly saccharide (LPS), a cytokine and any combination thereof.

Suitably, the method of the second, third, fourth, fifth and sixth aspects further includes the initial step of producing microglial cells from blood-derived cells isolated from the subject, such as that of the first mentioned aspect.

In a seventh aspect, the invention provides a kit for predicting the responsiveness of a neurodegenerative disease, disorder or condition to a therapeutic agent in a subject, the kit comprising at least one reagent capable of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from blood-derived cells isolated from the subject, wherein the level of phagocytosis, cytokine production and/or migration indicates or correlates with relatively increased or decreased responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent.

With respect to the second, third, fourth, fifth, sixth and seventh aspects, the microglial cells are suitably produced at least in part by culturing the blood-derived cells in the presence of interleukin-34 (IL-34) and granulocyte-macrophage colony stimulating factor (GM-CSF). Preferably, the microglial cells are produced at least in part by the method of the first aspect.

Suitably, the kit further comprises reference data for correlating the level of phagocytosis, cytokine production and/or migration and responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent. Preferably, the reference data is on a computer-readable medium.

In particular embodiments, the present kit is for use in the method of the second, third, fourth, fifth and sixth aspects.

With respect to the aforementioned aspects, the neurodegenerative disease, disorder or condition is or comprises Alzheimer’s disease, Parkinson’s disease or ALS.

In one embodiment, the blood-derived cell of the aforementioned aspects is or comprises a peripheral blood mononuclear cell (PBMC).

In an eighth aspect, the invention provides a microglial cell produced according to the method of the first aspect.

Suitably, the microglial cell is for use in the method or kit of the second, third, fourth, fifth, sixth and seventh aspects.

Suitably, the subject of the above aspects is a mammal, preferably a human.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Schematic of hiMG preparation technique. Monocytes were separated from a heterogeneous mixture of blood peripheral blood mononuclear cells (PBMCs) and cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL) - 34. These cytokines function as white blood cell growth factors and increase survival of monocytes, promoting differentiation to a microglia-like state. Blood was collected and added on top of a Ficoll-Paque layer in a SepMate-50ml tube to separate PBMCs from whole blood. Sample was centrifuged for 1200g for 10 minutes with deceleration at max allowing red blood cells to sediment and separate from PBMCs. PBMCs were tipped into a fresh Falcon tube. Two washes with phosphate buffer saline (PBS) were performed to allow the removal of any plasma that may have been collected during isolation of PBMCs. PBMCs were cultured with FBS, RPMI 1640 with GlutaMAX to provide cells with essential vitamins, proteins, lipids and growth factors. PBMCs were plated in designated cell culture plates or chamber slides and culture media (RPMI 1640 + GlutaMAX), supplemented with cytokines GM-CSF (lOng/ml) and IL-34 (lOOng/ml) was changed every 3 days to allow for differentiation into icroglia-like cells. Monocytes were differentiated into human induced microglia-like cells (hiMG) in the presence of GM-CSF and IL-34.

Figure 2. Phase contrast images of human monocyte-derived macrophages (A) and monocyte-derived microglia (hiMG) (B and C).

Figure 3. Images of hiMG labelled for microglia markers (P2ry 12, Tmeml 19 and

Cx3Crl) or macrophage marker (Ccr2). Arrows indicate cell nuclei.

Figure 4. Graphs of hiMG marker gene expression. Expression of genes at day 14 in vitro was determined by qRT-PCR relative to cultures at day 7.

Figure 5. Images of hiMG phagocytosis of fluorescently-labelled E.coli and amyloid peptide. A: uptake of red fluorescent E.coli into hiMGs over 6 hr in culture. B: Uptake of green fluorescently labelled amyloid beta peptide in hiMG, co-stained for microglia marker P2ryl2 (purple).

Figure 6. Graphs of cytokine gene expression in hiMGs. PAb, IL-6 and TNFa gene expression levels were determined in hiMGs stimulated with 20 ng/mL LPS by qRT- PCR relative to 18S mRNA expression (house-keeping gene). Fold change in gene expression was normalised to untreated samples. n=3. P-values were calculated using a t- test in GraphPad Prism.

Figure 7. Graph of Alzheimer’s patient and control hiMG TNFa expression. AD = Alzheimer’s disease, Ctr = matched controls ns = not significant. TNFa gene expression levels were determined by qRT-PCR relative to 18S mRNA expression (house-keeping gene). P-values were calculated using a t-test in GraphPad Prism.

Figure 8. Graph of Alzheimer’s patient and control hiMG IL-6 expression (ns = not significant). IL-6 gene expression levels were determined by qRT-PCR relative to 18S mRNA expression (house-keeping gene).

Figure 9. Graph of ALS patient and control hiMG IL-6 expression. A:

Comparison of age-matched controls, rapid, slow and intermediate disease cohorts. B: Comparison of individual patients with age-matched control cohort (ns = not significant). IL-6 gene expression levels were determined by qRT-PCR relative to 18S mRNA expression (house-keeping gene). P-values were calculated using a t-test in GraphPad Prism.

Figure 10. Graph of ALS patient and control hiMG TGF expression (ns = not significant). TGF gene expression levels were determined by qRT-PCR relative to 18S mRNA expression (house-keeping gene).

Figure 11. Graph of ALS patient and control hiMG IL-8 expression (ns = not significant). IL-8 gene expression levels were determined by qRT-PCR relative to 18S mRNA expression (house-keeping gene). P-values were calculated using a t-test in GraphPad Prism.

Figure 12. Graph of ALS patient and control hiMG IL-10 expression (ns = not significant). IL-10 gene expression levels were determined by qRT-PCR relative to 18S mRNA expression (house-keeping gene). P-values were calculated using a t-test in GraphPad Prism ns = not significant.

Figure 13. Graph of Alzheimer’s and control patient hiMG phagocytosis of fluorescently-labelled E.coli particles over 24 hours.

Figure 14. Graph of Alzheimer’s and control hiMG migration.

Figure 15. hiMG (green, Ibal detection) in 3D co-culture with ReN cell-derived neurons (purple, beta-tubulin detection).

Figure 16. A: hiMG (pink, small arrows, Ibal detection) in 3D co-culture with ReN cell-derived neurons (purple, beta-tubulin detection) and amyloid aggregate (green, large arrow). B: Measurement of fluorescent amyloid remaining after addition of hiMG (iMG). Demonstrates that the microglia are capable of removing (phagocytosing) the amyloid aggregates.

Figure 17. Human induced microglia exhibiting a ramified morphology when differentiated with ReN cells without the use of IL-34 and GM-CSF.

Figure 18. Human induced microglia cells and ReN cells were dissociated from matrigel matrix after 9 days of co-culture and were replated into a 3D OrganoPlate. Arborisation of microglia can be observed 24 hours after plating (arrows).

Figure 19. Effects of inflammatory -modulating drugs on Alzheimer’s disease microglia cytokine expression. A) Three Alzheimer’s patient hiMG cultures were exposed to the inflammatory modulating drugs, Dasatinib, Spiperone and Telmisartan and pro-inflammatory cytokine IL-6 expression was subsequently measured. B) Three Alzheimer’s patient hiMG cultures were exposed to the inflammatory modulating drugs, Dasatinib, Spiperone and Telmisartan and pro-inflammatory cytokine TNFa expression was subsequently measured. Dasatinib and Telmisartan both inhibited pro-inflammatory cytokine expression in all patients (IL-6) and some patients (TNFa). Spiperone increased cytokine expression. Individual Alzheimer’s patient hiMG responded differently to each of the drugs. *p=0.01, **0.001, ***0.0001, ****<0.0001.

Figure 20 provides graphical and photographic images showing improvements in hiMG cells. (A) Survival graph of 2D vs 3D hiMG culture; (B) Phase contrast and immunofluorescence image of hiMG in 2D and 3D; (C)Branch length and end-point measurement of microglia morphology in 2D vs 3D; (D) Microglial genes mRNA expression in 2D vs 3D; (E) Differential cytokine expression in 2D vs 3D; and (F) Validation of ReNVM and hiMG co-culture.

DETAILED DESCRIPTION

The present invention is at least partly predicated on the surprising discovery that the production of microglial cells from peripheral blood mononuclear cells by co-culture thereof with neural stem cells (NSC) not only serves as an experimental model that more closely mimics the in vivo brain microenvironment, but also significantly enhances survival of the microglial cells therein. Additionally, the invention described herein is predicated on the discovery that such microglial cells are useful in diagnosing, treating and/or determining the responsiveness to treatment and/or prognosis of neurodegenerative diseases, disorders and conditions, and in particular, Alzheimer’s disease, Parkinson’s disease and ALS.

Methods of Preparing Microglial Cells

In a broad form, the present invention relates to in vitro methods of producing a microglial cell from a blood-derived cell, and more particularly a monocyte, obtained or otherwise derived from a subject. In some embodiments, the blood-derived cell presents at least one cell surface marker selected from the group comprising or consisting of: CD2, CDl lb, CDl lc, CD 14, CD16, CD31, CD56, CD62L, CD115, CD192, CX3CR1, CXCR3, and CXCR4. In some preferred embodiments, the blood-derived cell is a monocyte. Suitably, the blood-derived cell may be derived or harvested from any biological sample of the patient, and particularly from samples containing significant levels of monocytes, such as blood, bone marrow, and cerebrospinal fluid (CSF). In some embodiments, the monocyte is obtained from a preparation of peripheral blood mononuclear cells (PBMCs).

In one particular aspect of this form, the present invention resides in a method of producing a microglial cell from a blood-derived cell of a subject, the method including the step of culturing the blood-derived cell with or in the presence of one or a plurality of neural stem cells to thereby produce the microglial cell. Preferably, the blood-derived cell is a monocyte.

The term“ microglial celF or“ microglia”, as generally used herein, refers to a class of glial cells involved in the mediation of an immune response within the central nervous system by generally acting as macrophages. Microglial cells are typically capable of producing exosomes, and further include different forms of microglial cells, including amoeboid microglial cells, ramified microglial cells and reactive microglial cells. Reactive microglia are generally defined as quiescent ramified microglia that transform into a reactive, macrophage-like state and accumulate at sites of brain injury and inflammation to assist in tissue repair and neural regeneration if required.

In this regard, microglia have long been implicated in the pathogenesis of various neurodegenerative diseases, disorders or conditions, such as those hereinafter described. The specific cellular actions of microglia as it relates to such neurodegenerative diseases, disorders or conditions, however, is poorly understood owing to the dearth of suitable in situ models of such cells (see, e.g., US2016/0333316). In view of this, the present invention relates to a novel technique of generating induced microglial (iMG) cells from blood-derived cells, including monocytes. By culturing blood-derived cells, and more particularly monocytes, in the absence of cytokines IL-34 and GM-CSF together with neural stem cells (NSC), these are converted into iMG cells within about 9 days (compared with the 14 days required for the traditional IL-34/GM-CSF methods described in US2016/0333316). The iMG cells described herein also express microglial markers, form a ramified morphology, and demonstrate phagocytic activity which is accompanied with cytokine releases, as per microglial cells. Suitable microglial markers include, but are not limited to, AIF1 and CD4.

The terms“ blood cells’’ or“ blood-derived cells’’ and the like, as used herein refer to cells or cell groups which may be present in the bloodstream in vivo , but not necessarily limited to peripheral blood cells. In this regard, the blood-derived cells may also be found or be able to be isolated from a cell population from, for example, bone marrow or umbilical cord blood, as well as pleural, peritoneal, cerebrospinal or synovial fluids or from various tissues, such as spleen and lymph node. In specific embodiments, the blood- derived cells express one or more marker molecules of blood cells, such as CD45, CD1 la.

In one embodiment, the blood-derived cells are or comprise blood-derived mononuclear cells, such as peripheral blood mononuclear cells (PBMCs).

The terms“ peripheral blood mononuclear celF or“ PBMC relate to a peripheral blood cell having a round nucleus. These cells typically include lymphocytes (T cells, B cells, NK cells) and monocytes, whereas erythrocytes and platelets have no nuclei, and granulocytes (neutrophils, basophils, and eosinophils) have multi-lobed nuclei. These cells can be extracted from whole blood using Ficoll and gradient centrifugation, which will separate the blood into a top layer of plasma, followed by a layer of PBMCs and a bottom fraction of polymorphonuclear cells (such as neutrophils and eosinophils) and erythrocytes, as hereinafter described.

In an alternative embodiment, the blood-derived cells are bone marrow-derived mononuclear cells.

In preferred embodiments, the blood-derived cells are monocytes.

As used herein, the term“ neural stem celF (NSC) or“ neural progenitor celF refers to a stem or progenitor cell typically found in adult neural tissue that can give rise to neurons and glial (supporting) cells. Examples of glial cells include astrocytes and oligodendrocytes. In some embodiments, the NSC or neural progenitor cell is an immortalised human neural stem cell line. In some other embodiments, the NSC or neural progenitor cell is an immortalised human neural progenitor cell line. In some embodiments, the NSC or neural progenitor cell is derived from the cortical region of human brain. In some alternative embodiments, the NSC or neural progenitor cell is derived from the ventral mesencephalic (VM) region of the brain.

In some embodiments, the NSC or neural progenitor cell is immortalized using retroviral transduction with the myc oncogene. Such methods are well known in the art, such as that described in Donato et ah, 2007). Non-limiting examples of stable NSC lines suitable for use in the methods of the present invention include ReNcell™ cells (e.g., ReNcell™ CX cells or ReNcell™ VM cells). ReNcell™ cell lines have been shown to propagate perpetually in culture and exhibit properties of human NSCs, including expression of NESTIN in an undifferentiated state and differentiation into specific cell types, including neuronal and glial cells, following deprivation of growth factors in culture medium. In another embodiment, the neural stem cells are or comprise embryonic stem cell-derived neural stem cells. To this end, it will be appreciated that the NSCs described herein can be derived from embryonic stem cells (ESCs), which can be utilised as pluripotent cells to provide an essentially unlimited and renewable source of NSCs. A number of protocols known in the art have been developed to differentiate ESCs into expandable NSC populations, and to derive potentially functional neurons and glial cells in a controlled manner (see, e.g., Hovakimyan et ah, (2012), Ann Anat. 194: 429-435; Woo et ah, (2009) BMC Neurosci. 10: 97; Breunig et al., (2011) Neuron. 70:614-625).

Suitably, the method of the present aspect further includes the initial step of isolating the blood-derived cell from the subject. Specifically, in some embodiments the methods include the step of isolating monocytes from the subject. This may be achieved by any method known in the art, such as Ficoll medium and gradient centrifugation, fluorescence activated cell sorting (FACS) and magnetic beads cell sorting (MACS). Suitably, monocytes are positively selected from a heterogenous population of cells by the use of one or more cell surface markers, selected from the group comprising CD2, CDl lb, CDl lc, CD 14, CD16, CD31, CD56, CD62L, CD115, CD192, CX3CR1, CXCR3, and CXCR4. Other methods can include the isolation of monocytes by depletion of non-monocytes cells (negative selection).

Suitably, the method of the present aspect further includes the subsequent step of collecting, harvesting or isolating the microglial cell. To this end, the microglial cell may be collected from the culture by any means known in the art, such as flow cytometry or magnetic cell sorting. Any cell surface marker known to be suitable for isolating or enriching microglial cells from a heterogenous cell sample, including but not limited to P2ryl2, Cx3Crl, Tmeml 19, Iba-1, and Trem 2, are applicable for use in the methods of the present invention. Once collected, the microglial cells can then be used in one or more of the hereinafter described aspects.

For the purposes of this invention, by“isolated' is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

The method of culturing the monocytes in the culture medium may be performed according to standard methods for culturing animal or mammalian cells, particularly microglial cells. In this regard, any culture medium known in the art, including natural culture medium or a synthetic culture medium, can be used as the culture medium. In this regard, the culture medium for co-culturing monocytes with the NSC may be a DMEM culture medium. Examples of suitable commercially available DMEM culture medium include DMEM/F12 (comprising 1 : 1 mixture of DMEM and Ham’s F-12 nutrient mixture) culture medium; DMEM RPMI-1640 culture medium, and a combination thereof. In some particularly preferred embodiments, the culture medium comprises, consists, or consists essentially of approximately 1 : 1 ration of DMEM/F12 medium and DMEM RPMI-1640 medium. Preferably, the culture medium is supplemented with GlutaMAX™ Supplement (i.e., L-alanyl-L-glutamine dipeptide in 0.85% NaCl). Typically, the culture media is supplemented with a concentration range of GlutaMAX Supplement of between about 0.1 mM to about 2 mM, and even more preferably between about 0.25 mM and about 1 mM. In some embodiments, the culture media comprises GlutaMAX Supplement at a concentration of about 0.5 mM.

Typically, the culturing is performed at a temperature between about 25° C and about 40° C. More specifically, the culturing is generally performed at a temperature between about 35° C and about 38° C. Even more specifically, culturing is performed at a temperature of between about 36.5° C and about 37.5° C. By way of an illustrative example, culturing may suitably be performed at about 37° C.

Typically, culturing is performed for between 1 day and 30 days (preferably, between 10 and 15 days).

Furthermore, culturing typically occurs at between about 2% CO2 and 10% CO2. In some particularly preferred embodiments, culturing occurs at about 5% CO2.

During culturing, the culture may be supplemented by one or more antibiotic such as ampicillin or penicillin as required.

Generation of 3-Dimensional Cell Cultures

Advantageously, co-culturing monocytes with NSCs in a three-dimensional (3D) cell culture system results in an extended survival time of the resultant microglial cells, to at least several months. In contrast, microglial cells produced using conventional two- dimensional (2D) culture methods typically have a survival time of approximately three weeks. The present technology therefore provides a highly supportive 3D environment for induced microglial cell (iMG) co-culture with neurons and astrocytes to examine patient microglia function in a brain-like environment. Accordingly, in some embodiments, the invention provides methods of generating an induced microglial cell (iMG) from a blood-derived cell (preferably, a monocyte) in a 3D culture system. In embodiments of this type monocytes are co-cultured with NSCs, using the culture methods described above and/or elsewhere herein (optionally without the addition of exogenous IL-34 and/or GM-CSF).

In some of the same embodiments and some alternative embodiments the iMGs are derived from monocytes cultured with or in the presence of one or both of IL-34 and GM-CSF. Such cytokines are commercially available and may be added to a culture medium separately, simultaneously, or in the form of mixture/cocktail of such cytokines. Suitably, the concentration of GM-CSF in the culture medium may range from about 1 ng/ml to about 200 ng/mL (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,

45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180

190, 200 ng/mL and any range therein). In some preferred embodiments, the concentration of GM-CSF in the culture medium is between about 1 ng/mL to about 50 ng/mL, and even more preferably between about 5 ng/mL to about 20 ng/mL.

The concentration of IL-34 in the culture medium may range from about 1 ng/mL to about 200 ng/mL (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 ng/mL and any range therein). Preferably, the concentration of IL-34 in the culture medium may range between about 50 ng/mL and about 150 ng/mL, and in some particular embodiments between about 80 ng/mL and about 120 ng/mL.

In particular embodiments of this type, the neural stem cells may be present as one or more 3D structures such as neural, cerebral or brain organoids. By“organoid” is meant an organized mass of cell types generated in vitro that mirrors (at least to some degree) the structure, marker expression, or function of a naturally occurring organ. The brain organoids may be incorporated within a scaffold, such as a polyester fleece or biodegradable polymer scaffold, to thereby produce a neural 3D structure.

The terms“ cerebral organoid ",“ neural organoicT and“ brain organoicT, as used interchangeably herein, refer to an artificial three-dimensional tissue culture created by culturing neural stem cells in, for example, a three-dimensional rotational bioreactor. Brain organoids may be synthesized tissues that contain several types of nerve cells and have anatomical features that can resemble mammalian brains. For example, brain organoids can comprise a heterogenous population of cells of at least two different progenitor and neuronal differentiation layers. Brain organoids may display heterogeneous regionalization of various brain regions as well as development of complex, well-organized cerebral cortex.

In one particular embodiment, the blood-derived cell and the neural stem cells are cultured, at least in part, in a 3D co-culture system.

Co-culture refers to the culture of more than one cell line (such as more than one of the disclosed cell lines), more than one cell type, or a cell line and a tissue sample, such a sample of a tumour biopsy, in a single vessel. A 2-dimensional (2D) co-culture is a co culture wherein the different cell lines or cell types are not cultured within the same dimension or layer of the culture, and are separated for example, by a gelled layer of gel matrix. A 3-dimensional (3D) co-culture is a co-culture wherein the different cell lines or cell types are cultured together within a three-dimensional support structure. In some embodiments the support structure may be a gel matrix (e.g., a hydrogel matrix). In this regard, the gel matrix may be any as are known in the art. In some embodiments, the gel matrix is a commercially available medium such as BD Matrigel™ Matrix (BD Bioscience), Cultrex® BME (Trevigen), or Geltrex® (Invitrogen®). Other basement membrane extracts that can function as a support matrix scaffolding in 3D co-culture systems include human placenta-derived BME (Vivo Biosciences, Inc) and synthetic BME (available from Glycosan Biosystems).

In one preferred embodiment, the blood-derived cell is cultured with the one or plurality of NSC substantially in the absence of exogenous IL-34 and GM-CSF. In this regard, the present inventors have surprisingly discovered that microglial cells can be generated directly by co-culture of the blood-derived cells or monocytes with NSC, such as NSC in the form of a brain organoid, and without the need to add exogenous IL-34 or GM-CSF.

Without being bound by any theory, it is believed that neurons, astrocytes or both derived from the NSC produce an environment that is conducive for the production or induction of microglial cells from blood-derived cells, and in particular, monocytes. In some preferred embodiments, the NSC is cultured with the 3D support matrix for at least around 24, 48, 72, 96 or more hours prior to introducing the monocytes or iMG cells to the culture. In some embodiments of this type, monocytes are introduced to the NSC at a ratio of between around 1 :3 to about 1 :7.5 (monocytes NSC). Preferably, a ratio of monocytes to NSC of around 1 :5 is used. In some alternative embodiments, the generation of a brain organoid can be performed by introducing iMG directly to an NSC being cultured in a 3D support structure.

Induced Microglial (iMG) Cells

The present invention provides induced microglial (iMG) cells that differ from those prepared using prior art methods, and those that exist naturally. Substantial differences in cellular expression levels (including cell surface marker expression), morphology and size can each be used to identify cells prepared using the present technology.

By way of an illustrative example, the iMG cells of the present invention exhibit enhanced expression levels of GPR34, PROS1, and TREM2 than microglial cells prepared through conventional 2D culturing methods. Accordingly, in some embodiments the present invention provides an isolated induced microglial cell, wherein the cell expresses one or more of CPR34, PROS1, and Tmeml 19, and Trem 2.

In a related aspect, the invention provides a microglial cell produced by the method of any one of the aforementioned aspects.

Methods of Diasnosins Neurodesenerative Disease

In certain embodiments, the microglial cells produced by the methods of the present aspects are for use in the methods hereinafter described.

In another aspect, the invention resides in a method of diagnosing a neurodegenerative disease, disorder or condition in a subject, including the step of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from monocytes isolated from the subject, wherein the level of phagocytosis, cytokine production, and/or migration is altered or modulated in the microglial cells. In some embodiments, the level of phagocytosis, cytokine production, and/or migration is altered or modulated as compared to a reference or control level.

In the context of the present invention, by“a disease, disorder or condition” is meant any disease, disorder and/or condition that comprises a progressive decline and/or deterioration in the structure, function, signalling and/or population of the neurons or neural tissue in an animal. In particular embodiments, the neurodegenerative disease, disorder or condition is or comprises a neuromuscular disease, disorder or condition. As used herein,“a neuromuscular disease, disorder or condition” refers to any disease, disorder and/or condition that comprises a progressive decline and/or deterioration in the structure, function, signalling and/or population of the neurons or neural tissue that innervate and/or communicate, whether directly or indirectly, with the muscles of an animal.

The aetiology of a neurodegenerative disease, disorder or condition may involve, but is not limited to, inflammation, genetic mutations, protein misfolding and/or aggregation, autoimmune disorders, mitochondrial dysfunction, defective axonal transport, aberrant apoptosis and/or autophagy and elevated oxidative stress and/or reactive oxygen species (ROS) production.

Without limitation, neurodegenerative diseases, disorders or conditions include Parkinson’s disease and related disorders, Huntington’s disease, Alzheimer’s disease and other forms of dementia, Spinocerebellar ataxia, Friedreich ataxia, Tay-Sachs disease, Lewy body disease, Parkinson’s disease and related disorders, Prion diseases (e.g. Creutzfeldt-Jakob disease), Multiple sclerosis (MS), Pick disease, Shy-Drager syndrome, pontocerebellar hypoplasia, neuronal ceroid lipofuscinoses, Gaucher disease, neurodegeneration with brain iron accumulation, spastic ataxia/paraplegia, supranuclear palsy, mesolimbocortical dementia, thalamic degeneration, cortical-striatal-spinal degeneration, cortical -basal ganglionic degeneration, cerebrocerebellar degeneration, Leigh syndrome, post-polio syndrome, hereditary muscular atrophy, encephalitis, neuritis, hydrocephalus and the motor neurone diseases.

In the context of the present invention, the subject with a neurodegenerative disease, disorder or condition may be undergoing a treatment regimen (preventative and/or therapeutic). In embodiments of this type, the subject may have been determined to either (i) have an existing neurodegenerative disease, disorder or condition; or (ii) be predisposed to such a disease, disorder or condition.

In one embodiment, the neurodegenerative disease, disorder or condition of this broad aspect is a motor neurone disease (MND).

Broadly, MNDs are a form of neurodegenerative diseases that typically involve the motor neurons of an affected subject. As would be readily understood by a skilled artisan, motor neurons are nerve cells that control the voluntary muscles of the trunk, limbs and phalanges, as well as those muscles that influence speech, swallowing and respiration. Accordingly, the clinical symptoms of a MND may include muscle weakness and/or wasting, muscle cramps, dysphagia, slurred speech, muscle tremors/fasciculations, reduced cognition, dyspnoea, respiratory failure, fatigue and weight loss without limitation thereto. MND includes, but is not limited to, amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig’s disease), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbar palsy and spinal muscular atrophy (SMA).

In light of the foregoing, the MND may be ALS, PLS, PMA, PBP, pseudobulbar palsy or SMA. MND neuron disease is ALS.

In particular preferred embodiments, the neurodegenerative disease, disorder or condition is or comprises Alzheimer’s disease.

In other particularly preferred embodiments, the neurodegenerative disease, disorder or condition is or comprises Parkinson’s disease.

In other particularly preferred embodiments, the neurodegenerative disease, disorder or condition is or comprises ALS.

The term “ determining’’ includes any form of measurement, and includes determining if an element is present or not. As used herein, the terms“ determining’ “ measuring’’,“ evaluating’ “assessing and“assaying" are used interchangeably and include quantitative and qualitative determinations. Determining may be relative or absolute.“ Determining the presence of’ includes determining the amount of something present ( e.g. , a cytokine biomarker), and/or determining whether it is present or absent.

As would be understood by the skilled person, the level of phagocytosis, cytokine production and/or migration of the microglial cells is deemed to be “ altered’ or “ modulated’ when the amount or level thereof is increased or up regulated or decreased or down regulated, as defined herein.

In one embodiment, the neurodegenerative disease, disorder or condition is detected if the level of phagocytosis, cytokine production and/or migration of the microglial cells are at a reduced level, down regulated or absent. In an alternative embodiment, the neurodegenerative disease, disorder or condition is detected if the level of phagocytosis, cytokine production and/or migration of the microglial cells are at an increased level, up regulated or present.

By way of an example, the neurodegenerative disease, disorder or condition is detected on the determination of the level of one or more proinflammatory cytokine being increased or up regulated. Suitable proinflammatory cytokines in this regard include, but are not limited to, TNF-a, IL-6, IL-Ib, and IL-8.

In some of the same embodiments and some other embodiments, the neurodegenerative disease, disorder or condition is detected if the level of one or more protective cytokines (e.g., TGF and/or IL-10) is decreased or down-regulated. By“ enhanced ",“ increased” or“ up regulated” as used herein to describe the level of phagocytosis, cytokine production and/or migration of the microglial cells, refers to the increase in and/or amount or level of level of phagocytosis, cytokine production and/or migration of the microglial cells when compared to a control or reference sample (e.g., control microglial cells derived from healthy individuals) or further microglial cells derived from a further biological sample from a subject. The level of phagocytosis, cytokine production and/or migration may be relative or absolute. In some embodiments, the level of phagocytosis, cytokine production and/or migration is more than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400% or at least about 500% higher than that level observed in a control sample or further biological sample from a subject (e.g., control microglial cells; or microglial cells obtained or otherwise derived from a healthy subject).

The terms,“ reduced” and“ down regulated as used herein to describe the level of phagocytosis, cytokine production and/or migration of the microglial cells, refer to a reduction in and/or amount or level thereof when compared to a control or reference sample (e.g., control microglial cells derived from healthy individuals) or further biological sample (e.g., further microglial cells) from a subject. The level of phagocytosis, cytokine production and/or migration may be relative or absolute. In some embodiments, the level of phagocytosis, cytokine production and/or migration is reduced or down regulated if its level is more than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, or even less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or 0.0001% of the level thereof in a control sample or further biological sample from a subject (e.g., control microglial cells; or microglial cells obtained or otherwise derived from a healthy subject).

The term“ control sample” typically refers to a biological sample from a healthy or non-diseased individual not having a neurodegenerative disease, disorder or condition (e.g., control microglial cells). In one embodiment, the control microglial cells may be derived from a subject known to be free of a neurodegenerative disease, disorder or condition. The control sample may be a pooled, average or an individual sample. An internal control is a marker from the same biological sample being tested.

In further embodiments, the level of phagocytosis, cytokine production and/or migration is compared to a predetermined threshold level thereof, such as a level of phagocytosis, cytokine production and/or migration observed in microglial cells derived from healthy or control subjects. Typically, a level of phagocytosis, cytokine production and/or migration of a microglial cells that exceeds or falls below the predetermined threshold level thereof is predictive of a particular disease state or outcome, such as the presence or absence of the disease, disorder or condition, or a resistance or responsiveness of the subject’s neurodegenerative disease, disorder or condition to a therapeutic agent. The nature and numerical value (if any) of the predetermined threshold level will typically vary based on the method chosen to determine the level of phagocytosis, cytokine production and/or migration, used in determining, for example, a diagnosis, a prognosis and/or a response to a therapeutic agent or treatment, in the subject.

A person of skill in the art would be capable of determining the predetermined threshold level of phagocytosis, cytokine production and/or migration of a microglial cell that may be used in determining, for example, a diagnosis, a prognosis and/or a response to a therapeutic agent, using any method of measuring phagocytosis, cytokine production and/or migration known in the art, such as those described herein. In one embodiment, the threshold level is a mean and/or median level (median or absolute) of phagocytosis, cytokine production and/or migration in a reference population of microglial cells, that, for example, have the same neurodegenerative disease, disorder or condition as said subject for which the level is determined. Additionally, the concept of a predetermined threshold level should not be limited to a single value or result. In this regard, a predetermined threshold level of phagocytosis, cytokine production and/or migration may encompass multiple predetermined threshold levels that could signify, for example, a high, medium, or low probability of, for example, response to a therapeutic agent, as described herein. Additionally, multiple threshold levels may assist in staging and/or grading particular neurodegenerative diseases, disorders or conditions.

As used herein, the term“ predetermined threshold’ refers to a value, above or below which, indicates the responsiveness of a disease to a treatment, or the general diagnosis or prognosis of the disease. For example, for the purposes of the present invention, a predetermined threshold may represent the level of phagocytosis, cytokine production and/or migration, in a sample from an appropriate control subject, such as a subject that is known to be healthy, or from multiple control subjects or medians or averages of multiple control subj ects. Thus, a level above or below the threshold indicates the likelihood of a subject having a neurodegenerative disease, disorder or condition, as taught herein. In other examples, a predetermined threshold may represent a value larger or smaller than the level determined for a control subject so as to incorporate further degree of confidence that a level or ratio above or below the predetermined threshold is indicative of disease, disorder or condition being present or absent in the subject. For example, the predetermined threshold may represent the average or median activity level of phagocytosis, cytokine production and/or migration in a group of control subjects, plus or minus 1, 2, 3 or more standard deviations. Those skilled in the art can readily determine an appropriate predetermined threshold based on analysis of microglial cells produced from monocytes obtained from appropriate control subjects. As used herein, the term “ phagocytosis” is a specific form of endocytosis involving the vesicular internalization of solid particles. It is therefore distinct from other forms of endocytosis such as pinocytosis, the vesicular internalization of various liquids, and receptor-dependent endocytosis. Phagocytosis is the process by which cells, such as microglia, ingest large objects or molecular aggregates, such as b-amyloid, a-synuclein, or TDP-43. The membrane folds around the object, and the object is sealed off into a large vacuole known as a phagosome. The phagosome is usually delivered to a lysosome, an organelle involved in the breakdown of cellular components, which fuses with the phagosome. The contents may then be degraded and either released extracellularly via exocytosis, or released intracellular to undergo further processing.

Assaying the level of phagocytosis of the microglial cell may be performed by any method known in the art. By way of example, methods known for assaying the phagocytic function of a phagocytic cell include exposing the phagocyte to latex particles, and determining the rate at which latex particles are engulfed in the cell by cytometry or microscopic observation. Alternatively, the phagocyte is exposed to foreign fluorescence-labelled substances (e.g., E. coli , zymosan, and the like), and the amount of the substance engulfed by the phagocyte is detected. An alternative method for assessing microglial phagocytosis include determining the number of viable bacteria by cultivation after the phagocytosis of the living bacteria, or determining luminescence from oxygen radical during the microglial phagocytosis.

Suitably, the neurodegenerative disease, disorder or condition is detected in a subject if the level of phagocytosis of the microglial cells is reduced as compared to the level of phagocytosis of the microglial cells from a subject that does not have a neurodegenerative disease, disorder or condition (e.g., a healthy control subject).

The cytokine to be measured for its production level can be any cytokine, including proinflammatory cytokines, that can be induced to be expressed and/or secreted from microglial cells. In certain embodiments, the cytokine is selected from the group consisting of TNFa, IL-Ib, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, TGFp, IFNy, fractalkine and any combination thereof. Evaluation of the production (e.g., expression and/or secretion thereof) of the cytokine can be by any means known in the art, including quantitation of expression levels of the cytokine, inclusive of RNA, mRNA, cDNA and protein levels (e.g., with ELISA, Western blot, qPCR and the like), or by bioassay, (e.g., determining whether cytokine activity is reduced). Migration of the microglial cells may be assessed by any means known in the art. Exemplary methods for studying cell migration include, for example, the Boyden Chamber Assay and the Scratch Wound Assay. The Boyden Chamber Assay generally involves placing cells on one side of a membrane. The membrane has pores of a diameter smaller than the diameter of the cells under investigation. After the cells are placed on one side of the membrane, the chamber is incubated for a period of time. Cell migration may be assessed by determining the number of cells that are present on the other side of the membrane after the period of time. The Scratch Wound Assay generally involves scraping a confluent monolayer of cells thereby creating a “wound” in the monolayer. Additionally, depositing cells in predetermined, defined, locations onto a substrate provides the ability to use video microscopic analysis of the serial motion, and hence migration, of the cells.

Suitably, the neurodegenerative disease, disorder or condition is detected in a subject if the level of migration of the microglial cells is reduced as compared to the level of migration of the microglial cells from a subject that does not have a neurodegenerative disease, disorder or condition (e.g., a healthy control subject).

In addition to the aforementioned assessments of the levels of phagocytosis, cytokine production and/or migration of the microglial cells, a number of additional cellular activities thereof may also be assessed in relation to the methods described herein. These may include, for example, proliferation capacity, viability, neurite elongation capability, morphological changes and/or differentiation capacity, albeit without limitation thereto. Again, these additional cellular activities may be performed by any means known in the art.

In a further aspect, the invention provides a method of determining a prognosis for a neurodegenerative disease, disorder or condition in a subject, said method including the step of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from monocytes isolated from the subject, wherein the level of phagocytosis, cytokine production and/or migration indicates or correlates with a less or more favourable prognosis for the neurodegenerative disease, disorder or condition.

The terms“ prognosis” and“ prognostic” are used herein to include making a prognosis, which can provide for predicting a clinical outcome (with or without medical treatment), selecting an appropriate course of treatment (or whether treatment would be effective) and/or monitoring a current treatment and potentially changing the treatment. This may be at least partly based on determining levels of phagocytosis, cytokine production and/or migration of the microglial cells, which may be in combination with or addition to determining the further cellular activities of the microglial cells, such as those hereinbefore described. A prognosis may also include a prediction, forecast or anticipation of any lasting or permanent physical or psychological effects of the neurodegenerative disease, disorder or condition suffered by the subject after the neurodegenerative disease, disorder or condition has been successfully treated or otherwise resolved. Furthermore, prognosis may include one or more of determining disease progression, including disease stage and/or grade, therapeutic responsiveness, implementing appropriate treatment regimes, determining the probability, likelihood or potential for disease recurrence after therapy and prediction of development of resistance to established therapies. It would be appreciated that a positive prognosis typically refers to a beneficial clinical outcome or outlook, such as long-term survival without recurrence of the subject’s neurodegenerative disease, disorder or condition, whereas a negative prognosis typically refers to a negative clinical outcome or outlook, such as disease recurrence or progression.

Suitably, the method of the present aspect further includes the step of diagnosing said subject as having a less favourable prognosis or a more favourable prognosis. In one embodiment, a relative or absolute increase in the levels of phagocytosis, cytokine production and/or migration of the microglial cells is diagnostic of a less favourable or poor prognosis in the subject. In a further embodiment, a relative or absolute decrease in the levels of phagocytosis, cytokine production and/or migration of the microglial cells is diagnostic of a more favourable prognosis in the subject.

For example, if the microglial cells of the subject demonstrated increased levels of phagocytosis, increased levels of migration and/or decreased levels of proinflammatory cytokine production this would indicate the subject having a more favourable prognosis. Conversely, if the microglial cells of the subject demonstrated decreased levels of phagocytosis, decreased levels of migration and/or increased levels of proinflammatory cytokine production this would indicate the subject having a less favourable prognosis.

With respect to the above, a neurodegenerative disease, disorder or condition may have a relatively poor prognosis due to one or more of a combination of features or factors including: at least partial resistance to therapies available for treatment thereof; advanced or end-stage disease; and a low probability of patient survival, although without limitation thereto.

It will also be understood that levels of phagocytosis, cytokine production and/or migration of the microglial cells may be used to identify those poorer prognosis patients, such as those with more aggressive, advanced or progressive neurodegenerative diseases, disorders or conditions, who may benefit from one or more additional therapeutic agents to the typical or standard treatment regime for that particular patient group.

Suitably, the neurodegenerative disease, disorder or condition is any known in the art, such as those hereinbefore provided. In particular preferred embodiments, the neurodegenerative disease, disorder or condition is or comprises Alzheimer’s disease, Parkinson’s disease, or ALS.

Assessing Responsiveness to Therapeutic Agents

It will be appreciated from the hereinafter that the invention provides methods of using the microglial cells and/or organoids generated using the methods described above and/or elsewhere herein to determine the responsiveness of a neurodegenerative disease, disorder or condition to a candidate therapeutic agent. The therapeutic agent may be a composition that is already known to treat a subset of subjects with a neurodegenerative disease, disorder, or condition, or alternatively, could be a new candidate therapeutic agent that is not currently commercially available for the treatment of a neurodegenerative disease, disorder or condition. Particular broad embodiments of the invention include the step of treating the patient following predicting a positive responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent. Accordingly, these embodiments relate to using information obtained about the predicted responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent to thereby construct and implement a treatment regime for the patient. In a preferred embodiment, this is personalized to a particular patient so that the treatment regime is optimized for that particular patient.

Accordingly, in one aspect the invention provides a method of predicting the responsiveness of a neurodegenerative disease, disorder or condition to a therapeutic agent in a subject, said method including the step of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from monocytes isolated or otherwise obtained from the subject, wherein the level of phagocytosis, cytokine production and/or migration indicates or correlates with relatively increased or decreased responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent. In some embodiments, the responsiveness of a subject’s neurodegenerative disease, disorder or condition to the therapeutic agent is increased relative to a subject that is not considered to respond to the therapeutic agent. In some alternative embodiments, the responsiveness of the subject with a neurodegenerative disease, disorder or condition to the therapeutic agent is decreased relative to a subject that is considered to positively respond to the therapeutic agent.

The term“ therapeutic agent” as generally used herein refers to a chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject (such as the improvement in one or more signs or symptoms of a neurodegenerative disease, disorder or condition), alone or in combination with another therapeutic agent(s) or pharmaceutically acceptable carriers. Exemplary therapeutic agents include, but are not limited to, anti-psychotics (e.g., spiperone), flavonoids (e.g., diosmin), Src inhibitors (e.g., dasatinib), antihypertensives, such as angiotensin II receptor blockers (e.g., telmisartan), antioxidants (e.g., resveratrol, ginsenoside), iron chelators (e.g., deferoxamine), anti-inflammatories, including NS AIDs (e.g., naproxen), antibiotics including tetracyclines (e.g., minocycline), copper- containing compounds, bis(thiosemicarbazones), and hydroxyquinolines.

In view of the above, the present method may further include the step of treating the neurodegenerative disease, disorder or condition in the subject. By way of example, this can include administering to the subject a therapeutically effective amount of the therapeutic agent when the level of phagocytosis, cytokine production and/or migration indicates or correlates with relatively increased responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent.

As used herein, the term“therapeutically effective amount” describes a quantity of a specified agent (e.g., a therapeutic agent), sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of a composition comprising one or more agents that are necessary to reduce, alleviate and/or prevent a neurodegenerative disease, disorder or condition. In some embodiments, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of a neurodegenerative disease, disorder or condition. In other embodiments, a “therapeutically effective amount” is an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease or prevent disease progression or overcome resistance to and/or enhance the therapeutic activity of a therapeutic agent.

In a preferred embodiment, the therapeutic agent or treatment is administered when the levels of phagocytosis, cytokine production and/or migration of the microglial cells indicates or correlates with relatively increased responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent.

In some embodiments, the present method further includes the step of contacting the microglial cells with the therapeutic agent. To this end, the therapeutic agent may be administered to the subject in question when the levels of phagocytosis, cytokine production and/or migration of the subject’s microglial cells are shown to be favourably altered or modulated upon contact with the therapeutic agent to as to provide an indication of responsiveness of the subject with a neurodegenerative disease, disorder or condition to the therapeutic agent.

By way of example, if the subject’s microglial cells are exposed to a candidate therapeutic agent and shown to exhibit increased phagocytosis and/or migration compared with those levels thereof prior to contact of the microglial cell with the candidate therapeutic agent (i.e., baseline levels) or those levels of a microglial cell that is not exposed to the candidate therapeutic agent (i.e., control levels), the candidate therapeutic agent may be considered to be an enhancer of microglial activity for the subject. To this end, the subject’s neurodegenerative disease, disorder or condition may be considered to be responsive to said candidate therapeutic agent. For example, the candidate therapeutic agent may be selected as a therapeutic agent suitable for promoting the removal of b-amyloid, a-synuclein, or TDP-43, or the like present in the subject’s brain.

Furthermore, if the subject’s microglial cells upon contact with a candidate therapeutic agent are shown to exhibit decreased proinflammatory cytokine production (e.g., TNF-a) compared with those levels thereof prior to contact of the microglial cell with the candidate therapeutic agent (i.e., baseline levels) or those levels of a microglial cell that has not been placed in contact with the candidate therapeutic agent (i.e., control levels), the candidate therapeutic agent may be considered to be an inhibitor of the inflammatory activity for the microglial cells of the subject. Again, the subject’s neurodegenerative disease, disorder or condition may be considered to be responsive to said candidate therapeutic agent. For example, the candidate therapeutic agent may be selected as an anti-inflammatory agent.

It will be appreciated that the method of the present aspect may further include the step of contacting the microglial cells with a stimulant, such as an immune stimulant. To this end, the contacting step may be performed before, during and/or after contacting the microglial cells with the therapeutic agent so as to assess whether the therapeutic agent may be suitable for treating existing disease and/or inflammation and/or preventing subsequent disease and/or inflammation from occurring.

The stimulant may be any as are known in the art. In particular embodiments, the stimulant is selected from the group consisting of a b-amyloid peptide, a-synuclein, a tau protein, TDP-43, a lipopolysaccharide (LPS), a cytokine, SOD1, and any combination thereof. In one preferred embodiment, the stimulant is or comprises a Damage-associated molecular pattern (DAMP).

In a related aspect, the invention resides in a method of treating or preventing a neurodegenerative disease, disorder or condition in a subject, the method including the step of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from monocytes isolated from the subject and based on the determination made, initiating, continuing, modifying or discontinuing administration of a therapeutic agent to the subject.

As used herein,“ treating’’ (or“ treat” or“ treatment’) refers to a therapeutic intervention that ameliorates a sign or symptom of a neurodegenerative disease, disorder or condition after it has begun to develop. A“ prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a neurodegenerative disease, disorder or condition, or exhibits only early signs, for the purpose of decreasing the risk of developing a symptom, aspect, or characteristic of a neurodegenerative, neuropsychiatric and/or neuromuscular disease, disorder or condition. A“ therapeutic” treatment is one administered to a subject who exhibits at least one symptom, aspect, or characteristic of the neurodegenerative, neuropsychiatric and/or neuromuscular disease, disorder or condition so as to cure, remediate or reverse, at least in part, and/or halt or delay the progression of said symptom, aspect, or characteristic.

As used herein,“ preventing’’ (or“ prevent’ or“ preventative”) refer to a course of action (such as administering a therapeutic agent demonstrating therapeutic potential when contacted with the subject’s microglial cells) initiated prior to the onset of a symptom, aspect, or characteristic of the neurodegenerative disease, disorder or condition so as to prevent said symptom, aspect, or characteristic. It is to be understood that such preventing need not be absolute to be beneficial to a subject.

In one embodiment, the present method further includes the step of contacting the microglial cells with the therapeutic agent.

Suitably, the present method further includes the step of contacting the microglial cells with a stimulant. Preferably, the stimulant is an immune stimulant, such as a Damage-associated molecular pattern (DAMP). In particular embodiments, the stimulant is selected from the group consisting of a beta amyloid peptide, a-synuclein, a tau protein, TDP-43, SOD1, a lipopolysaccharide (LPS), a cytokine and any combination thereof.

It will be appreciated that the therapeutic agent may be any known in the art, such as those hereinbefore described.

Additionally, the neurodegenerative disease, disorder or condition is suitably any known in the art, such as those hereinbefore provided. In particular preferred embodiments, the neurodegenerative disease, disorder or condition is or comprises Alzheimer’s disease, Parkinson’s disease or ALS.

In another related aspect, the invention provides a method of stratifying a subject having a neurodegenerative disease, disorder or condition for a clinical trial of a therapeutic agent including the steps of:

In the case of prospective trials, detection of the levels of phagocytosis, cytokine production and/or migration from the microglia of prospective patients may be used to stratify patients prior to their entry or inclusion into the trial or while they are enrolled in the trial. In clinical research, stratification is the process or result of describing or separating a patient population into more homogeneous subpopulations according to specified criteria. Stratifying patients initially rather than after the completion of a trial is frequently preferred, e.g., by regulatory agencies such as the U.S. Food and Drug Administration that may be involved in the approval process for a therapeutic agent. In some cases, patient stratification may be required by the study design. It will be appreciated that further stratification criteria may be employed in conjunction with determining levels of phagocytosis, cytokine production and/or migration of microglial cells. Commonly used criteria include age, family history, disease stage and/or grade, etc. Stratification is frequently useful in performing statistical analysis of the results of a trial.

Suitably, the present method further includes the step of contacting the microglial cells with a stimulant. Preferably, the stimulant is an immune stimulant, such as a Damage-associated molecular pattern (DAMP). In particular embodiments, the stimulant is selected from the group consisting of a beta amyloid peptide, a-synuclein a tau protein, TDP-43, SOD1, a lipopolysaccharide (LPS), a cytokine and any combination thereof.

Further, the neurodegenerative disease, disorder or condition may be any known in the art, such as those hereinbefore provided. In particular preferred embodiments, the neurodegenerative disease, disorder or condition is or comprises Alzheimer’s disease, Parkinson’s disease, or ALS.

Kits

In yet another aspect the invention provides a kit for predicting the responsiveness of a neurodegenerative disease, disorder or condition to a therapeutic agent in a subject, the kit comprising at least one reagent capable of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from blood-derived cells isolated from the subject, wherein the level of phagocytosis, cytokine production and/or migration indicates or correlates with relatively increased or decreased responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent.

Suitably, the microglial cells are produced at least in part by culturing the blood- derived cells with or in the presence of IL-34 and GM-CSF. Alternatively, the microglial cells are preferably produced according to the method of the first mentioned aspect.

In certain embodiments, the kit further comprises reference data for correlating the level of phagocytosis, cytokine production and/or migration with responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent.

In particular embodiments, the reference data is on a computer-readable medium (e.g., software embodying or utilised by any one or more of the methodologies or functions described herein). The computer-readable medium can be included on a storage device, such as a computer memory (e.g., hard disk drives or solid state drives) and preferably comprises computer readable code components that when selectively executed by a processor implements one or more aspects of the present invention.

Suitably, the present kit is for use in the method of the aforementioned aspects.

In a particular embodiment, the present kit provides a“ companion diagnostic’’ whereby information with respect to phagocytosis, cytokine production and/or migration of microglial cells are utilized by a clinician or similar for the safe and effective administration of a therapeutic agent.

Suitably, the neurodegenerative disease, disorder or condition is any known in the art, such as those hereinbefore provided. In particular preferred embodiments, the neurodegenerative disease, disorder or condition is or comprises Alzheimer’s disease or ALS.

With respect to the aforementioned aspects, the term“ subject” includes but is not limited to mammals inclusive of humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs). Preferably, the subject is a human.

Unless the context requires otherwise, the terms“ comprise”,“ comprises” and “ comprising or similar terms are intended to mean a non-exclusive inclusion, such that a recited list of elements or features does not include those stated or listed elements solely, but may include other elements or features that are not listed or stated.

The indefinite articles‘a’ and‘ an’ are used here to refer to or encompass singular or plural elements or features and should not be taken as meaning or defining“one” or a “single” element or feature. For example,“a” cell includes one cell, one or more cells and a plurality of cells.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference. The following non-limiting examples illustrate the methods and kit of the invention. These examples should not be construed as limiting: the examples are included for the purposes of illustration only.

EXAMPLE 1

Monocvte-derived microglia-like cells provide a rapid, cost-effective approach to patient-specific screening of neuroimmune modulatory drugs for neurodegenerative diseases: An alternative approach using microglia-like cells has recently been developed. [12]-[15] This advanced approached involves the rapid induction of human blood-derived monocytes into microglia-like cells. The adult brain is populated by resident microglia that migrated to the brain during early development. However, during disease blood Monocyte-Derived microglia (MD-microglia) are also localized to the brain. The exact role of MD-microglia in neurodegenerative disease brain is not well understood, but they are believed to have an important role in aggregated protein clearance. [16]-[ 17] It remains extremely challenging to distinguish between resident and MD-microglia, and this provides a major advantage for using MD-microglia as a model for identifying therapeutic drugs for patient microglia. It would be anticipated that beneficial drugs identified using MD-microglia should target both resident and MD- microglia in a patient’s brain. [15] To generate MD-microglia in vitro , monocytes are isolated from peripheral blood mononuclear cells (PBMCs) collected from patient blood by standard Ficoll-Paque density gradient centrifugation. [12]-[15] Monocytes are grown in culture media containing granulocyte-macrophage colony-stimulating factor (GM- CSF) and the cytokine interleukin 34 (IL-34) for 2 weeks. [12]-[14] The cells transform from predominantly small, round monocytes into highly ramified cells with the morphology of mature resting microglia (human induced microglia. hiMG) (Fig. 2). [12]- [14]

The hiMG reveal high expression of microglia-selective markers, together with down-regulated expression of monocyte markers as assessed by gene expression, flow cytometry, and immunofluorescence analysis. [12]-[15] The hiMG also have microglial- like phagocytic activity (and increased expression of microglia phagocytosis genes), and release cytokines in response to inflammatory stimulation as expected for mature microglia. [12]-[15] hiMG reveal a remarkably close relationship to mature human microglia in terms of cell surface marker expression, function, and gene expression. [12]- [15]

Generating hiMG is rapid (2 weeks) and cost-effective (~AUD$250/person, including labour) compared to iPSC-derived microglia (months and up to AUD$ 10,000/person). iMG generation can therefore potentially be applied to patient blood samples to produce‘real-time’ data on patient microglia immune responses and screen for prospective immuno-modulatory therapeutic compounds. This is a major step towards individualized patient immuno-therapy. Furthermore, the rapid and cost-effective process would allow the continuous monitoring of drug effectiveness in‘at-risk’ or diagnosed neurodegenerative disease patients through sequential blood sampling. This would allow real-time adjustment of immune-modulation therapy. Real-time cost- effective screening of patient microglia would also provide critical information for selection and stratification of patients and monitoring efficacy of compounds in clinical trials.

METHODS

1. GENERATION OF hiMG FROM MONOCYTES.

Plasma collection from EDTA-blood tubes

1. Spin down blood tubes at 300g for 5 mins

2. Collect 500m1 of plasma in an Eppendorf tube, without touching the blood layer.

If that is not possible, collect 200-300m1.

3. Label and store plasma at -80°C

4. Replace the same amount of plasma removed with PBS + ImM EDTA (RT).

5. Mix blood sample and continue with Ficoll separation below.

PBMC isolation using SepMate50

Human PBMCs were purified from whole blood using Ficoll-Paque Plus (1.077 ± 0.001 g/ml density, GE Healthcare, Sweden) density gradient centrifugation (Beckman Coulter Allegra xl5-R), using one SepMate-50ml tube (StemCell Technologies) at the speed of 1200g for 10 min with deceleration max (Figure 1). The buffy coat was tipped into a fresh tube and washed twice with Phosphate buffer saline (PBS) with ImM Ethyl enediaminetetraacetic acid (EDTA) (PBS: 8.5g/L NaCl, 1.48 g/L dibasic Sodium phosphate, 0.43g/L monobasic Potassium Phosphate) at 300g for 10 mins with maximum deceleration. The supernatant was aseptically removed, and the pellet was resuspended in freezing media consisting of 90% fetal bovine serum (FBS) + 10% dimethyl sulfoxide (DMSO). A cell count was done by using a hemocytometer in 1/10 dilution with trypan blue. Cells that were stained blue indicating dead cells were excluded. Cells were then frozen at -80°C in Mr. Frosty (Thermo Scientific) and transferred later for liquid nitrogen storage.

Thawing of PBMCs

For preparing frozen human PBMCs for hiMG differentiation, PBMCs were thawed rapidly in a 37°C water bath and diluted dropwise with 9ml pre-warmed media containing 90% RPMI 1640 + Glutamax containing 1% P/S (Penicillin/Streptomycin) and 10% FBS. The cell suspension was centrifuged at 300g for 5 min, after which supernatant was decanted. Cell pellet was resuspended in pre-warmed media and plated into the appropriate culture vessel as described below.

Differentiation and maintenance of hiMG and monocyte derived macrophages (MDM)

Firstly, culture plates were coated with 1 : 100 Matrigel (Corning) in PBS at 37°C one day prior to use. To plate PBMCs, cells were resuspended in pre-warmed media ((RPMI 1640 + GlutaMax (Gibco, Life Technologies) containing 10% heat-inactivated FBS and 1% Penicillin Streptomycin (P/S). Cells were counted and plated at various densities depending on the experimental approach. As an experimental control, PBMCs were also differentiated into monocyte-derived macrophages (MDM). On day 2 following cell plating, media was changed to RPMI 1640 + Glutamax, 1% P/S + lOOng/ml IL-34 (Lonza) and lOng/ml GM-CSF (Lonza) for hiMG differentiation or to RPMI 1640 + Glutamax, 10% FBS, 1% P/S and lOng/ml GM-CSF for MDM differentiation. Cells were cultured in standard conditions (37°C, 5% CO2) (Thermo-Fischer forma steri-cycle C02 incubator). To maintain the cells, half media changes were performed every 3 days for up to 14 days, after which cells were utilised for downstream analysis.

Differentiation of monocytes into ramified human microglia-like cells (hiMGs)

Selection of cytokines to induce PBMCs into hiMG was based on previous findings by Ohgidani et al , who have reported GM-CSF and IL-34 as essential cytokines for developing and maintaining hiMG.[13] GM-CSF is a white-blood cell growth factor and stimulates monocytes to mature macrophages. IL-34 is a ligand for cerebrospinal fluid receptor 1 (CSF1R) and found to be a key cytokine in development of microglia and maintaining them in a steady state. Monocytes induced by 10 ng/ml GM-CSF and 100 ng/ml IL-34 to generate hiMG showed small circular morphology on the day of exposure to cytokines, however differentiation into elongated structures was noticed by day 6 - day 9 in culture. By day 12, cells exhibited small and large branches originating from the soma and a ramified microglia-like branched morphology was achieved by 14 days in culture (Figures 2 and 3). When differentiating PBMCs into MDM, it was observed that untreated monocytes preserved small circular shape in the presence of RPMI + GlutaMAX and 10% FBS (Figure 2).

2. CHARACTERIZATION OF hiMG.

a. Microglia-specific markers

hiMG exhibit characteristic marker expression of resident brain microglia

To demonstrate that the hiMG revealed expected expression of microglia- associated cell markers we performed immunofluorescence with fluorescently-labelled antibodies to different microglia (or macrophage) markers. As shown in Figure 3, hiMGs (CD45^low cells) revealed strong staining for microglia markers P2ryl2, Cx3Crl, and Tmeml 19, but not the macrophage marker CCR2. Figure 4 reveals increased expression of these markers (and microglia markers Iba-1 and Trem2) in hiMG cells at day 14 compared to day 7 but similar changes were not seen in MDMs (CD45^Mgh cells).

b. Phagocytosis

An important function of microglia is phagocytosis (uptake and digestion of unwanted material). To examine this, we cultured hiMGs with fluorescently-labelled E.coli (bacteria) (red in Figure 5) for up to 6 hours. Figure 5 shows increasing red fluorescence associated with hiMGs over time, demonstrating strong phagocytic activity by most cells. Similarly, when hiMG were cultured with fluorescently-labelled amyloid peptide, the cells phagocytosed the amyloid as evidenced by the green fluorescence associated with the cells (Figure 5). These findings confirm that hiMGs perform microglial-like phagocytosis.

c. Cytokine response

Another important measure of microglial behaviour is cytokine expression and release in response to inflammatory stimulation. As shown in Figure 6, hiMGs treated with the classical inflammatory molecule, lipopolysaccharide (LPS) revealed up- regulation of pro-inflammatory cytokine expression (IL-Ib, IL-6, and TNFa). This confirms that hiMGs can respond to stimulation as expected for microglia. 3. IDENTIFYING ALZHEIMER’S AND ALS PATIENT-SPECIFIC DIFFERENCES IN hiMG RESPONSES.

a. Cytokine responses

hiMGs derived from patient PBMCs have a strong potential to identify patient- specific differences in microglial function and gene expression on a clinically relevant scale. The hiMGs can also offer a potential platform for patient-specific drug screening, or stratification of patients for clinical trials of new drugs. Patient-specific differences in hiMGs may also contribute to diagnostic processes, particularly in people suspected of dementia.

To determine if there are patient-specific differences in hiMGs from people with Alzheimer’s disease, we have examined cytokine release from cultured patient and matched control hiMGs (Figure 7). Patient and control hiMGs were generated and cultured as above and cytokine levels measured by qPCR for gene expression. As shown in Figure 7, healthy control hiMGs revealed low levels of the pro-inflammatory cytokine, TNFa. In contrast, Alzheimer’s disease patient hiMGs revealed high levels of TNFa (ADI, AD3), moderate levels (AD4) or low levels (AD2, AD5). As indicated in the accompanying table, there was a significant difference in the TNFa levels between most of the AD patient hiMGs, but not between the controls. These findings demonstrate that the patient-derived hiMGs can be used to identify not only disease-associated changes compared to controls groups, but also differences in microglial responses between individual patients with the same disease. These differences could be used as a basis for diagnosis, stratification for clinical trials, or selection of specific drugs to treat individual patients.

2. Inflammatory cytokine IL-6.

As shown in Figure 8, a similar outcome was observed for the pro-inflammatory cytokine, interleukin-6 (IL-6) in Alzheimer’s disease patients. Age-matched control hiMGs revealed low levels of IL-6 with little variation between people. In contrast, of 5 Alzheimer’s patient hiMGs examined, 3 showed similar expression levels of IL-6 to the control group, but 2 patients revealed significantly higher IL-6 levels (Figure 8), demonstrating again individual patient differences in inflammatory response of hiMGs.

We also examined IL-6 expression in hiMGs derived from PBMCs of people with amyotrophic lateral sclerosis (ALS), the most common form of motor neuron disease. ALS patients were divided into those with rapidly progressing disease (rapid), slowly progressing disease (slow), or intermediately progressing disease (intermediate) (Figure 9). As shown in Figure 9, there was a significant difference in hiMG IL-6 expression between ALS patient levels with rapid or slow disease, compared to matched controls. As with Alzheimer’s patients, several ALS patients revealed significantly higher levels of IL-6 compared to the control group, while other patients showed no significant change, demonstrating patient-specific hiMG inflammatory responses.

3. Inflammatory cytokine TGFp

Analogous outcomes were observed when transforming growth factor beta (TGFP) expression levels were measured in ALS patient hiMGs. As seen in Figure 10, control patients revealed consistently low level TGFp levels, while all rapid disease ALS patients and one of three slow disease ALS patients revealed significantly elevated TGFp expression in hiMGs. This again demonstrated patient-specific expression of cytokines in hiMGs.

4. Inflammatory cytokine IL-8

A similar but less distinct result was observed when interleukin-8 (IL-8) expression levels were measured in ALS patient hiMGs. As seen in Figure 11, control patients revealed consistently moderate levels of IL-8 expression, while most rapid and slow disease ALS patient hiMGs revealed slightly but not significantly lower IL-8 levels. In contrast, hiMGs from one patient with rapid disease revealed significantly elevated IL- 8 levels, demonstrating patient-specific expression of this cytokine in hiMGs.

5. Inflammatory cytokine IL-10

A similar observation was observed when we measured interleukin-10. Low expression levels were observed in control hiMGs and all ALS patient hiMGs except for one patient (Figure 12). Notably, the patient with high IL-10 levels in Figure 12 is not the same patient as the one with high IL-8 levels in Figure 11.

Phagocytosis

To determine whether Alzheimer’s disease patient hiMGs revealed differences in their phagocytic ability, we measured phagocytosis of fluorescent E.coli particles over 24 hrs. Figure 13 demonstrates the continuous uptake of E.coli particles as a measure against time and the graph of uptake over time reveals that age matched controls and Alzheimer’s patient hiMGs are significantly different. While there were no differences between each of the control patient hiMGs over 24 hours, each of the two Alzheimer’s patient hiMGs examined revealed different phagocytic activity between 12 and 24 hours (Figure 13). These findings provide further support for patient specific differences between hiMGs. hiMG migration

Another important function of microglia in the brain is the ability to migrate towards a target area to undertake an inflammatory response or repair tissue. We can measure this in cell culture by measuring the migration rate of hiMGs. As shown in Figure 14, we have observed that there are more hiMGs in patient AD2 than ADI that move at a velocity between 0.2 and 0.3 pixels/min. This shows that hiMGs from different patients can move at different speeds representing patient-specific differences.

4. DEMONSTRATION OF PATIENT-SPECIFIC DRUG RESPONSES USING hiMG.

Figure 19 demonstrates that 2D cultures of Alzheimer’s patient-derived hiMGs respond differently to several different drugs. In particular, the expression levels of IL-6 and TNFa cytokines was modulated differently between the three Alzheimer’s patient hiMG cultures assessed when exposed to the inflammatory modulating drugs of dasatinib, spiperone and telmisartan. This supports our view that these hiMGs can be used to screen for individual patient responses to drugs.

5. EXAMINATION OF hiMG FUNCTION AND DRUG ACTION IN 3D CO CULTURE.

a. hiMG and ReN neural stem cells in 3D

To generate a model of patient microglia function that more closely mimics the in vivo brain microenvironment we have added hiMGs to 3D co-cultures with a human neural stem cell line (ReN cell). The ReN cell line differentiates into a combination of neurons and astrocytes when growth factors are withdrawn from the culture medium. We have grown these cells in 3D using a 3D extracellular matrix (Matrigel™). To these cells has been added hiMGs (from normal healthy people) and we have found that the co culture environment provides excellent support for the hiMGs (Figure 15). hiMGs grown in 2D or 3D without co-culture only survive approximately 3 weeks. hiMGs grown in 2D co-culture with ReN cells also only survive for about 3 weeks. In contrast we have found that co-culture of hiMGs with ReN cells in 3D Matrigel results in extended survival to at least several months. This demonstrates that we have a highly supportive 3D environment for hiMG co-culture with neurons and astrocytes to examine patient microglia function in a brain-like environment. We have modelled the Alzheimer’s brain further by adding in pre-aggregated amyloid peptide (fluorescently labelled) and have shown that hiMGs can target these amyloid plaque-like structures in 3D culture (Figure 18). This will allow us to investigate patient-specific differences in Alzheimer’s hiMG interaction with amyloid plaque-like structures and the effect of potential therapeutics in a 3D microenvironment.

We have previously described in Example 1 a method of differentiating monocytes to microglia-like cells using growth factors such as IL-34 and GM-CSF for 14 days. This novel technique described below suggests that co-culturing monocytes with ReN cells containing neurons and astrocytes, can induce differentiation of microglia without the use of IL-34 and GM-CSF. This unique approach adds value to our understating of microglial function within the brain and is a new paradigm for modelling brain architecture and drug development.

ReN cell differentiation for co-culture

Immortalised ReNcells VM (ReN) are human neural progenitor cells (hNPCs) derived from the ventral mescencephalon region of the human foetal brain supplied commercially from Merck Millipore. ReN cells has the ability to differentiate into neurons and astrocytes cells upon growth factor deprivation.

Firstly, T75 flask were coated with 1/100 Matrigel diluted in DMEM/F12 + Glutamax for 1 hour at room temperature. Matrigel solution was removed before ReN cells were plated. Briefly, ReN cells were then thawed in basal medium containing DMEM/F12 + Glutamax + 2% B-27 supplement and 1% Penicillin Streptomycin (P/S) and spun down at 300g for 5 minutes. Centrifuged cells were decanted and resuspended in 10 mL of ReN cell basal medium supplemented with growth factors supplemented with b-Fibroblast Growth Factor (FGF-2; 20 ng/mL) and Epidermal Growth Factor (EGF; 20 ng/mL) (Lonza) and plated in matrigel coated flask. When 90% confluency was achieved, Accutase solution was used to detach ReN cells for 3 minutes at 37° C. To deactivate Accutase, basal medium with growth factors was added. Cells were centrifuged at 300 g for 5 minutes and replated at a density of 200,000 cells per well in a 6-well plate. To initiate spontaneous differentiation, FGF and EGF were redrawn from media. ReN cells will be differentiated for 1 month to allow the differentiation of mature neurons and astrocytes.

Preparation of monocytes for co-culture with ReN cells PBMCs were thawed and plated in a 6-well ultra-low attachment plate at 6 million cells per well with media containing RPMI 1640 + GlutaMax (Gibco, Life Technologies), 10% heat-inactivated FBS and 1% Penicillin Streptomycin (P/S). The following day, monocytes that are adhered on plastic were removed with existing media and centrifuged at 300 g for 5 minutes. Centrifuged cells were decanted and resuspended in ReN cell basal media containing 1/10 Matrigel before plating. 100,000 PBMCs were used for co-culture with differentiated ReN cells as described above for 9 days.

Human induced microglia and ReN cells can be detached from MATRIGEL matrix by using Cell Recovery solution (Corning). Briefly, conditioned media was saved for later use. 2 mL of ice cold PBS was used to rinse well before 1 mL of cell recovery solution was added. After 5 minutes of incubation, MATRIGEL that contains hiMG are collected in previously saved conditioned media and centrifuge at 300 g for 5 minutes. We have demonstrated that hiMG are both viable and preserves their ramified morphology. (Figures 17 and 18).

hiMG cultured in 3D exhibit longer survival times

Human induced microglia cultured in 2D or 3D were observed for their survival (days in culture). A significant increase in survival of hiMG grown in 3D was observed (i.e., 14 days ± 2 in the 2D cell culture system, as compared to 30 days ±10 in the 3D cell culture system) (Figure 20A) This suggests that the 3D culture system was able to provide a suitable microenvironment for the extended survival of hiMG.

Furthermore, phase contrast picture of hiMG grown in the 3D culture system showed increased complex arborisations compared to hiMG cultured in 2D system by day 14 in culture (see, Figure 20B). HiMG was confirmed as a brain microglia cell type by immunofluorescence technique, where the presence of microglia specific markers (such as P2ryl2 and Ibal) was observed (coloured greenin Figure 2B). Staining of the cell nucleus is labelled in blue (DAPI).

Accordingly, branch length and end-points (number of junctions) were analysed in hiMG cultured in 2D and 3D. A significant increased in branch length and end-points were observed in hiMG cultured in 3D than compared to 2D system (see Figure 20B). This confirms that although both 2D and 3D platforms were able to generate hiMG, the hiMG cultured in 3D platform are more representative of a human brain microglia.

A significant increase in microglial specific genes (such as Prosl, Gpr34,

Tmeml 19, and Trem2) was observed in cells obtained from the 3D cell culture system, while a decrease in myeloid gene, CD45 was noted. This suggests that hiMG cultured in the 3D system is more mature than compared to hiMG cultured in 2D system. Microglia specific genes have been published in Ryan et al 2017.

Materials & Methods

An mRNA expression study was performed by Real-Time PCR. In brief, hiMG were cultured and harvested at day 14 ± 2 in 2D cultures, while at day 30 ± 10 in 3D cultures. RNA were extracted and 10 ng of total RNA was used to convert to cDNA. cDNA was diluted at 1 : 10 with ultra pure water and used at 1 :4 (cDNA: Sybr green master mix) per reaction. Real-time PCR was performed using the Viia7 Real-time PCR machine. Annealing temperature of specific primer pairs were optimised at 58° C- 62° C.

Assays for Measuring Candidate Drug Response

Inflammation involves the release of cytokines that can be categorised to two main types: pro-inflammatory cytokines that promote inflammation and anti-inflammatory cytokines, which aids in resolving or attenuating inflammation. Accordingly, a patient with neurodegenerative disease, disorder or condition would have increased production of one or more pro-inflammatory cytokines (e.g., IL-6, TNF-a, IL-8, IL-Ib, and IL-18), while lower levels of one or more anti-inflammatory cytokines (e.g., IL-10, TGF ) The hiMG assays as described in the patent aim to determine patient cytokine levels and to predict the response to a candidate therapeutic agent by examining the cytokine levels of the subject.

3D co-culture involves the combination of two cell types (NSC and hiMG). To examine cell-specific response within a co-culture, isolation of hiMG can be done by FACs sort or magnetic isolation. Specific microglia markers used include CD45,

CD1 lb, Tmeml 19, and P2ryl2.

The overall aim of utilising a 3D cell culture platform is to provide a brain-like microenvironment that represents a brain milieu (e.g., a brain organoid). Hence, we it was next investigates whether cytokine response levels were similar or different in 3D cell culture systems compared to 2D cell culture systems. This in turn will help us understand how hiMG platform is relevant for drug testing/prediction.

Overall, we found significant differences in pro-inflammatory cytokine mRNA gene expression (e.g., TNFa, IL-8) and anti-inflammatory (e.g., IL-10) in hiMG cultured in 3D compared to 2D. This indicates that drug testing in widely used 2D culture system may differ when tested in a 3D platform. Immunofluorescence image of 3D co-culture shows the presence of NSC as identified with b3 tublin (TUBB, in red) and hiMG (Ibal, in green). The positive staining of TUBB indicates the presence of neurons, and the positive staining of Ibal indicates the presence of microglia. Overall confirming the suitable culture conditions of both cell types in 3D system.

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Claims

1. A method of producing a microglial cell from a monocyte, the method including the step of culturing the monocyte with one or a plurality of neural stem cells to thereby produce the microglial cell.

2. The method of Claim 1 , further including the initial step of isolating the monocyte from a subject.

3. The method of Claim 1 or Claim 2, further including the subsequent step of collecting/harvesting the microglial cell.

4. The method of any one of the preceding claims, wherein the neural stem cells are or comprise immortalized or stable neural stem cells and/or embryonic stem cell-derived neural stem cells.

5. The method of any one of the preceding claims, wherein the blood-derived cell and the neural stem cells are cultured, at least in part, in a 3-dimensional co-culture system.

6. The method of any one of the preceding claims, wherein the neural stem cells are or comprise a brain organoid.

7. The method of any one of the preceding claims, wherein the blood-derived cell is cultured with the one or plurality of neural stem cells substantially in the absence of interleukin-34 (IL-34) and granulocyte-macrophage colony stimulating factor (GM- CSF).

8. A method of diagnosing a neurodegenerative disease, disorder or condition in a subject, including the step of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from blood-derived cells isolated from the subject, wherein the level of phagocytosis, cytokine production and/or migration is altered or modulated in the microglial cells.

9. A method of determining a prognosis for a neurodegenerative disease, disorder or condition in a subject, said method including the step of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from blood-derived cells isolated from the subject, wherein the level of phagocytosis, cytokine production and/or migration indicates or correlates with a less or more favourable prognosis for the neurodegenerative disease, disorder or condition.

10. The method of Claim 9, wherein if the level of phagocytosis, cytokine production and/or migration is altered or modulated in the microglial cells, the prognosis may be negative or positive.

11. The method of Claims 9 or 10, wherein the prognosis is used, at least in part, to develop a treatment strategy for the subject.

12. The method of any one of Claims 9 to 11, wherein the prognosis is used, at least in part, to determine disease progression in the subject.

13. The method of any one of Claims 9 to 12, further including the step of determining suitability of the subject for treatment with a therapeutic agent based, at least in part, on the prognosis.

14. The method of any one of Claims 8 to 13, further including the step of determining a disease stage and/or grade for the neurodegenerative disease, disorder or condition based on, at least in part, the level of phagocytosis, cytokine production and/or migration.

15. A method of predicting the responsiveness of a neurodegenerative disease, disorder or condition to a therapeutic agent in a subject, said method including the step of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from monocytes isolated from the subject, wherein the level of phagocytosis, cytokine production and/or migration indicates or correlates with relatively increased or decreased responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent.

16. The method of Claim 15, further including the step of administering to the subj ect a therapeutically effective amount of the therapeutic agent when the level of phagocytosis, cytokine production and/or migration indicates or correlates with relatively increased responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent.

17. A method of treating a neurodegenerative disease, disorder or condition in a subject, the method including the step of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from blood-derived cells isolated from the subject and based on the determination made, initiating, continuing, modifying or discontinuing administration of a therapeutic agent to the subject.

18. A method of stratifying a subject having a neurodegenerative disease, disorder or condition for a clinical trial of a therapeutic agent including the steps of:

determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from blood-derived cells isolated from the subject; and, stratifying the subject for the clinical trial based on the results of the determining step.

19. The method of any one of Claims 15 to 18, further including the step of contacting the microglial cells with the therapeutic agent.

20. The method of any one of Claims 8 to 19, further including the step of contacting the microglial cells with a stimulant.

21. The method of Claim 20, wherein the stimulant is an immune stimulant.

22. The method of Claim 21, wherein the immune stimulant is or comprises a Damage-associated molecular pattern (DAMP).

23. The method of any one of Claims 20 to 22, wherein the stimulant is selected from the group consisting of a beta amyloid peptide, a-synuclein a tau protein, TDP-43, SOD1, a lipopolysaccharide (LPS), a cytokine and any combination thereof.

24. The method of any one of Claims 8 to 23, further including the initial step of producing microglial cells from blood-derived cells isolated from the subject.

25. A kit for predicting the responsiveness of a neurodegenerative disease, disorder or condition to a therapeutic agent in a subject, the kit comprising at least one reagent capable of determining a level of phagocytosis, cytokine production and/or migration of microglial cells produced from blood-derived cells isolated from the subject, wherein the level of phagocytosis, cytokine production and/or migration indicates or correlates with relatively increased or decreased responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent.

26. The method of Claim 24 or the kit of Claim 25, wherein the microglial cells are produced at least in part by culturing the blood-derived cells in the presence of interleukin-34 (IL-34) and granulocyte-macrophage colony stimulating factor (GM- CSF).

27. The method or kit of any one of Claims 22 to 23, wherein the microglial cells are produced at least in part by the method of any one of Claims 1 to 7.

28. The kit of any one of Claims 25 to 27, further comprising reference data for correlating the level of phagocytosis, cytokine production and/or migration and responsiveness of the neurodegenerative disease, disorder or condition to the therapeutic agent.

29. The kit of Claim 28, wherein the reference data is on a computer-readable medium.

30. The kit of any one of Claims 25 to 29, for use in the method of any one of Claims 8 to 24, 26 and 27.

31. The method or kit of any one of the preceding claims, wherein the neurodegenerative disease, disorder or condition is or comprises Alzheimer’s disease, Parkinson’s disease, or ALS.

32. The method or kit of any one of the preceding claims, wherein the subject is a human.

33. The method or kit of any one of the preceding claims, wherein the blood-derived cell is or comprises a preparation of peripheral blood mononuclear cells (PBMC).

34. The method or kit of any one of the preceding claims, wherein the blood-derived cell is a monocyte.

35. A microglial cell produced according to any one of Claims 1 to 7.

36. The microglial cell of Claim 34, for use in the method or kit of any one of Claims 8 to 33.

37. An isolated microglial cell, wherein the cell expresses any one of CD45, CD1 lb, Tmeml l9, and P2ryl2.

38. The isolated cell of claim 37, wherein the cell is derived from a monocyte.

39. The isolated cell of claim 37 or 38, wherein the cell comprises the cell surface marker selected from CD45, CD1 lb, Tmeml 19, and P2ryl2.

40. An organoid that comprises one or more microglial cells of any one of claims 37 to 39.