WO2018200669A1

WO2018200669A1 - Inducing microglia from hematopoietic and pluripotent stem cells

Info

Publication number: WO2018200669A1
Application number: PCT/US2018/029354
Authority: WO
Inventors: Derrick Rossi; Bruna PAULSEN; Manavi NAGAI
Original assignee: Boston Childrens Hospital
Current assignee: Boston Childrens Hospital
Priority date: 2017-04-25
Filing date: 2018-04-25
Publication date: 2018-11-01
Anticipated expiration: 2019-10-25

Abstract

Provided herein are robust and scalable methods of generating microglia-like cells (e.g., in vitro or ex vivo). The methods involve differentiating a cell (e.g., an iPS, a HSC, or a CD34+ cell) into a monocyte, and differentiating the monocyte into a microglia-like cell. In some embodiments, the methods further comprises expanding the cell prior to differentiation, thus enabling the generation of a large number of microglia-like cells from a smaller number of cells. The microglia-like cells generated using the methods described herein are homogenous and maintain the biological activities and functionalities of natural microglia cells.

Description

INDUCING MICROGLIA FROM HEMATOPOIETIC AND PLURIPOTENT STEM

CELLS

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/489985 filed April 25, 2017 and entitled "INDUCED MICROGLIA FROM HEMATOPOIETIC AND PLURIPOTENT STEM CELLS," the entire contents of which are incorporated by reference herein.

BACKGROUND

Microglia play an integral role in maintaining a healthy environment of the human brain and contribute greatly to the pathological effects of many neurological disorders.

However, various different aspects and functions of microglia remain unclear. To study cells like microglia from a human subject, either invasive procedures must be used to acquire live samples or research is constrained to post mortem observation.

SUMMARY

Described herein are methods of generating microglia-like cells from pluripotent stem cells (e.g., induced pluripotent stem cells or iPS cells) or multipotent stem cells (e.g., hematopoietic stem cells or HSCs) by differentiating the pluripotent stem cells or multipotent stem cells into monocytes, followed by differentiating the monocytes into microglia-like cells. In some embodiments, the methods described herein involve expanding the cell prior to differentiation, thereby allowing generating a large number of microglia-like cells from a small number of cells, or even from a single cell. The microglia-like cells generated using the methods described herein have typical functional and phenotypic characteristics as a natural microglia, e.g., having a ramified morphology, expressing microglia-like cell-specific markers, having phagocytic abilities, and/or secreting cytokines. The successful differentiation of HSCs into microglia with the efficient and scalable methods described herein enables various applications for the study of the multifaceted role of microglia in the human nervous system.

Accordingly, some aspects of the present disclosure provide methods of generating a microglia-like cell, the method including: (i) differentiating a cell that is an induced pluripotent cell (iPS), a hematopoietic stem cell (HSC), or a CD34+ cell into a monocyte; and (ii) differentiating the monocyte obtained in (i) into a microglia-like cell.

In some embodiments, the differentiating in (i) comprises contacting the cell with an effective amount of macrophage colony- stimulating factor (M-CSF), stem cell factor (hSCF), receptor-type tyrosine -protein kinase FLT3 (Flt-3), interleukin-3 (hIL-3), or combinations thereof. In some embodiments, the differentiating in (ii) comprises contacting the monocyte with an effective amount of granulocyte macrophage colony- stimulating factor (GM-CSF) and interleukin-34 (IL-34).

In some embodiments, the methods further includes expanding the cell prior to the differentiating of (i) to produce a population of the cell. In some embodiments, the expanding comprises contacting the cell with an effective amount of SCF, thrombopoietin (TPO), IL3, Flt3L, A83-01, Pomalidomide, and UM171.

In some embodiments, the cell is an iPS. In some embodiments, the cell is a HSC. In some embodiments, the HSC has a surface expression profile of lineage-CD235a- CD34+CD38-CD45RA-CD90+. In some embodiments, the cell is a CD34+ cell. In some embodiments, the CD34+ cell is generated from an iPS. In some embodiments, the CD34+ cell has a surface expression profile of CD34+CD45+.

In some embodiments, the monocyte expresses any one of CD14, CD16, CDl lb, and/or CD33. In some embodiments, the microglia-like cell expresses IBA1. In some embodiments, the microglia-like cell has an increased CX3CR1/CCR2 expression ratio compared to the monocyte. In some embodiments, the CX3CR1/CCR2 expression ratio is increased by at least 50% compared to the CX3CR1/CCR2 expression ratio of monocytes.

In some embodiments, the population of the cell is used to generate microglia-like cells. In some embodiments, at least 30% of the population of the cell are differentiated into monocytes after the differentiating in (i).

In some embodiments, the differentiating in (i) is carried out for no more thanl4 days. In some embodiments, the differentiating in (ii) is carried out for no more than 14 days. In some embodiments, the expanding is carried out for no more than 14 days.

Further provided herein are methods of method of generating a microglia-like cell, the method includes: (i) expanding a cell that is an induced human pluripotent cell (iPS), a hematopoietic stem cell (HSC), or a CD34+ cell to generate a population of the cell; (ii) differentiating the population of the cell obtained in (i) into monocytes; and (iii) differentiating the monocytes obtained in (ii) into microglia-like cells. In some embodiments, the method is carried out in vitro. In some embodiments, the method is carried out ex vivo. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

Other aspects of the present disclosure provide microglia-like cells generated by the methods described herein. In some embodiments, the microglia-like cell secretes a cytokine upon stimulation. In some embodiments, the cytokine is selected from the group consisting of: CCL2, MIP-la/b, CXCL10, IL-6, TNF-a, Serpin El, and combinations thereof. In some embodiments, the microglia-like cell is phagocytic. In some embodiments, the microglia-like cell expresses IBA1 on its surface. In some embodiments, the microglia-like cell has comparable biological activity as a natural microglia.

The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. In the drawings:

Figure 1. Protocol timeline. An overview of the timeline for HSC and CD34+ expansion and subsequent differentiation into microglia through a monocyte intermediate. Different media compositions, time points of analyses performed, and antibodies used for staining in FACS analysis are identified.

Figures 2A and 2B. Expansion of HSCs and CD34+ cells in vitro. Microscopy images of expanding HSCs and CD34+ cells in the various different expansion medias at day 5 (Figure 2A) and day 10 (Figure 2B). RM = Expansion Media, U = UM171 and S = SRI. Scale bar: lOOum.

Figures 3A-3D. Expansion media (RM) + UM171 is the most effective media for expanding HSCs and CD34+ cells while maintaining initial cell profile. (Figure 3A)

Representative FACS plots used to identify HSCs cells(Lin-CD34+CD38-CD45RA- CD90+CD49F+). (Figure 3B) Representative FACS plots used to identify CD34+ population (Lin-CD34+) (Figure 3C) Percentage of HSCs live (left column) and total HSC count (right column) found in the population of expanded HSCs starting from single, 5 and 25 cells.

(Figure 3D) Percentage of CD34+ cells live (left column) and total CD34+ cell count (right column) found in the population of expanded CD34+ cells starting from single, 5 and 25 cells. Significance was calculated using One-way ANOVA (*p<0.05, **p<0.01, ***p<0.001).

Figures 4A-4C. Induced microglia-like cells have ramified morphology. (Figure 4A) Representative microscopy images of control cells (maintained in Expansion media (RM) + UM171) and differentiating cells at days 27 - 42. (Figure 2B) Quantification of

morphological observation; the percentage of wells containing visible cells (left) and the percentage of wells that contained morphologically ramified microglia-like cells (right).

Figure 5. Clonal immunophenotypic HSC and CD34+ cells maintain their profile of markers after 14 days of expansion in conditions previously described.

Immunophenotypic HSC are defined here as CD34+Lin-CD45RA-CD90+CD49F+ and CD34+ as CD34+Lin-. Cells were analyzed by flow cytometry.

Figures 6A to 6B. Progression of changes in cell profile throughout the course of the expansion and differentiation protocol. (Figure 6A) Percent expression of markers CD34, CDl lb, CD33 and CD14CD16+ in control (maintained in expansion media) and differentiating cells throughout the protocol in sHSCs and sCD34 cells. (Figure 6B) Increase in expression ratio of CX3CR1/CCR2 throughout the course of the differentiation protocol as expected for induced microglia in comparison to monocytes. A higher ratio of

CX3CR1/CCR2 is characteristic of microglia and this ratio was observed to increase as the differentiation protocol progressed. By the end of the differentiation protocol, this ratio was more than twice as large as it was at the start of the monocyte stage.

Figures 7A-7D. Induced microglia-like cells show typical functional and phenotypic microglia characteristics. (Figure 7A) Upper panel: Immunocytochemistry of IBAl (red) and DAPI (blue) stained control cells (maintained in expansion media) and induced microglia-like cells from sHSCs and sCD34 cells. Lower panel: Higher magnification images of immunocytochemistry of IBAl (red) and DAPI (blue) shows characteristic morphology of induced microglia-like cells from sHSCs. (Figure 7B) Cells were incubated with GFP- fluorescent latex beads and imaged live after 24 hours in culture. Beads (green) can be observed co-localizing with cells. (Figure 7C) Immunocytochemistry performed with IBAl and DAPI following functional phagocytosis analysis. After the phagocytosis analysis was performed, cells were fixed and stained for IBAl (red) and DAPI (blue). GFP-fluorescent latex beads are shown in green, and can be seen co-localizing with cells as shown previously.

Overlaid image of phagocytosed beads (green), IBA1 (red) and DAPI (blue)

immunocytochemistry. (Figure 7D) Negative control for the immuno staining s. Scale bar A - C: lOOum, Scale bar B: 50um.

Figures 8A-8B Cytokine profile of induced microglia-like cells following endotoxin stimulation. (Figure 8A) Upper panel: Baseline cytokine expression of untreated microglia- like cells (top) and cytokine expression after treatment with IFN-gamma and LPS (bottom). Lower panel: Map of dots that were blotted, showing increases in expression from control to stimulated in red and a decrease in expression in blue. (Figure 8B) Left panel: Graphical representation of log2 of fold changes to demonstrate difference in expression of cytokines from stimulated and control cells. Right Panel: Raw fold changes between stimulated and control cells.

Figures 9A-9E. Differentiating CD34+ cells without expansion yields results similar to initial protocol of expansion followed by differentiation. (Figure 9A) Percent expression of markers CD34, CDl lb, CD33 and CD14+CD16 in differentiating sHSCs.

(Figure 9B) Immunocytochemistry of IBA1 (red) and DAPI (blue) of differentiating sHSCs (top). Results of phagocytosis assay performed on induced microglia-like cells from sHSCs show internalization of fluorescent latex beads (green) (bottom). (Figure 9C) Top panel: Baseline cytokine expression of untreated microglia-like cells (top) and treated with IFN- gamma and LPS (bottom). Lower panel: Map of dots that were blotted, showing increases in expression from control to stimulated in red. (Figure 9D) Raw fold changes between stimulated and control cells. (Figure 9E) Graphical representation of log2 of fold changes to demonstrate difference in expression of cytokines from stimulated and control cells.

Figures 10A-10E. Effects of JAK2 inhibitor TG101209 on the microglia-like cells. (Figure 10A) Cells were treated with JAK2 inhibitor TG101209, concomitant to treatment with LPS and IFN-γ for 24 hours. (Figure 10B) Corroboratively, application of TG 101209 on LPS and IFN-y treated microglia-like cells prevented an increase in the TNF-a concentration in the cells' supernatant. (Figures lOC-lOE) Analysis of treated cells following fixation and staining with IBA1 antibody showed detectable differences in cellular area (Figure. IOC), aspect ratio (Figure. 10D) and mean fluorescence intensity (MFI) (Figure. 10E).

Figures 11A-11B. Molecular profile of the microglia-like cells. (Figure HA)To further investigate the molecular signature of the microglia-like cells induced from HSPCs (CB CD34+ cells) and human iPSC-derived CD34+ cells (iPSC 1 and iPSC 2) compared to their parental cells and to primary human macrophages and microglia, the molecular profile of cells coming from each of the different sources was assessed. (Figure 11B) Differentiated cells clustered together with the primary microglia, while the macrophages were independently clustered, gathering nearer to the HSPCs. This data suggests that the transcription profile of induced microglia-like cells are closer to primary microglia than to their parental cells, macrophages, and/or monocytes.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

A "microglia" is a type of neuroglia located throughout the brain and spinal cord. A neuroglia (also termed "glial cell") is a non-neuronal cell that maintains homeostasis, forms myelin, and provides support and protection for neurons in the central and peripheral nervous systems. In the central nervous system, glial cells include oligodendrocytes, astrocytes, ependymal cells and microglia, and in the peripheral nervous system glial cells include Schwann cells and satellite cells. Naturally, microglial cells differentiate in the bone marrow from hematopoietic stem cells, the progenitors of all blood cells. During hematopoiesis, some of these stem cells differentiate into monocytes and travel from the bone marrow to the brain, where they settle and further differentiate into microglia.

Microglia account for 10-15% of all cells found within the brain. As the resident immune cell of the central nervous system, microglia play a crucial role in surveillance, pruning/neuromodulation and inflammation, among other functions in both the healthy and diseased brain (e.g., as described in Tremblay et al., 2011; Cartier et al., 2014, incorporated herein by reference). Formerly, microglia were thought to mostly serve a relatively singular role as key players in mediating brain injury and disease (e.g., as described in Cunningham et al., 2013, incorporated herein by reference). However, the role of microglia in the healthy brain remains unclear, though there is evidence suggesting that microglia could have potential roles in postnatal development, adult neuronal plasticity, and neuromodulation (e.g., as described in Tremblay et al, 2011, incorporated herein by reference). As such, microglial dysfunction could play crucial roles in neurological disorders such as, but not limited to, Alzheimer's disease, Parkinson's disease, multiple sclerosis, schizophrenia, prion disease, and age related dementia (e.g., as described in Cunningham et al., Glia 61:71-90; Vivekanantham et al., Int J Neurosci 5:717-25, 2015; Mosser et al., Progress in Neurobiology 150: 1-20, 2017, incorporated herein by reference). In order to study the role and biological functions of microglia, e.g., in neurological disease pathology, it is important to have a robust platform for studying microglia. To date, studying microglia (e.g., from a human subject) remains challenging because obtaining live microglia cells requires invasive procedures, or research is constrained to post mortem observation. Some aspects of the present disclosure are based, at least in part, on the findings that large numbers of microglia-like cells may be obtained by differentiating pluripotent stem cells (e.g., induced pluripotent stem cells or iPS), hematopoietic stem cells (HSCs), or CD34+ cells (e.g., CD34+ cells generated from iPS). Microglia cells generated using the methods described herein may be used in a platform to model healthy and diseased states of microglia. In addition, a variety of different drugs and chemical compounds can be tested on these microglia-like cells to identify therapeutic targets as well as drugs effective in treating microglial dysfunction.

Accordingly, some aspects of the present disclosure provide methods of generating microglia-like cells (e.g., in vitro). A "microglia-like cell" refers to a cell that resembles a microglia, e.g., in its morphology and biological function. For example, a microglia-like cell may adopt the ramified morphology (long branching processes and a small cellular body) of a microglia, and have comparable biological function (e.g., phagocytic function) of a microglia.

Methods of generating microglia-like cells have been described in the art. For example, Ohgidani et al., (Scientific reports 4:4957, 2014, incorporated herein by reference) developed a protocol to differentiate human monocytes, isolated from the peripheral blood, into microglia-like cells. However, this process requires a very large amount of cells, which limits the scalability of this protocol to be widely utilized as a platform to study microglial function and disease. In another example, Muffat et al., (Nat Med 22: 1358-1367, 2016, incorporated herein by reference) induced microglia-like cells from human induced pluripotent stem (iPS) and embryonic stem (ES) cells. Muffat et al. took advantage the differentiation potential of pluripotent stem cells to induce a yolk sac intermediate, which they subsequently differentiate into microglia-like cells. However, the method described in Muffat et al. is very laborious and requires complicated steps and takes close to 3 months.

In contrast, the methods of generating microglia-like cells described herein do not require more complex steps than culturing cells in differentiation media and take no more than 42 days or less. In some embodiments, the methods of the present disclosure may be used to generate a large number of microglia-like cells from very few number (e.g., less than 100, less than 10, or even a single cell) of cells (e.g., iPS, HSC, or CD34+cells). Thus, the methods described herein are more robust and/or scalable, and thus are superior to existing methods. The methods described herein may be used to generate large numbers of microglia-like cells, which in turn may enable a platform to model healthy and diseased states of microglia for studying microglia functions and interventions. In addition, a variety of different drugs and chemical compounds can be tested on these microglia-like cells to identify therapeutic targets as well as drugs effective in treating microglial dysfunction.

As described herein, the methods of generating microglia-like cells comprise (i) differentiating a cell that is an induced pluripotent cell (iPS), a hematopoietic stem cell (HSC), or a CD34+ cell into a monocyte; and (ii) differentiating the monocyte obtained in (i) into a microglia-like cell. "Differentiate" or "differentiation" refers to a process where a cell changes from one cell type (e.g., a pluripotent cell or a multipotent cell) to a more specialized cell type (e.g., a somatic cell).

A "monocyte" is a type of white blood cell, or leukocyte. Monocytes are the largest type of leukocyte and can differentiate into macrophages and myeloid lineage dendritic cells. As a part of the vertebrate innate immune system monocytes also influence the process of adaptive immunity. Monocytes compose 2% to 10% of all leukocytes in the human body and serve multiple roles in immune function. Such roles include, without limitation: replenishing resident macrophages under normal conditions; migration within approximately 8-12 hours in response to inflammation signals from sites of infection in the tissues; and differentiation into macrophages or dendritic cells to effect an immune response. In an adult human, half of the monocytes are stored in the spleen.

Cells that may be differentiated into monocytes include, without limitation, pluripotent cells (e.g., an iPS), a multipotent cell (e.g., a HSC), or a CD34+ cell (e.g., a CD34+ cell generated from an iPS). A pluripotent cell refers to a cell that has the potential to develop into more than one type of mature cell, depending on environment. A pluripotent cell can give rise to all cell types except for extra embryonic tissue. For example, a stem cell can differentiate into 3 germ layers i.e. endoderm, mesoderm, ectoderm. A pluripotent cell may be an embryonic stem cell (ES cell) or an induced pluripotent stem cell (iPS cell). An embryonic stem cell is a pluripotent stem cell derived from the inner cell mass of a blastocyst, an early- stage preimplantation embryo.

Accordingly, some aspects of the present disclosure relate to differentiating a cell (e.g., an iPS cell, a HSC, or a CD34+ cell) into a monocyte. In some embodiments, an iPS is differentiated into a monocyte. An "induced pluripotent stem (iPS) cell" refers to a type of pluripotent stem cell that is reprogrammed from a somatic cell (any cell of a living organism other than the reproductive cells) to a pluripotent state via different techniques, e.g., the introduction of four specific genes encoding transcription factors. In some embodiments, a HSC is differentiated into a monocyte. A "hematopoietic stem cell (HSC)" is a stem cell that gives rise to all the other blood cells through the process of hematopoiesis. Blood cells that may arise from HSCs include, without limitation, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., natural killer cells, B-cells, and T-cells). A HSC may be derived (i.e., differentiated) from a pluripotent stem cell (e.g., an embryonic stem cell or an induced pluripotent stem cell) or a cord blood stem cell.

HSCs are characterized by the absence of lineage- specific markers and expression of a cell surface molecule termed cluster of differentiation 34 (CD34). "CD34" is a protein belong to a family of single-pass transmembrane sialomucin proteins that show expression on early hematopoietic and vascular-associated tissue (e.g., as described in Neilson et al., Journal of Cell Science. 121 (Pt 22): 3683-92, 2008, incorporated herein by reference). CD34 is an important adhesion molecule and is required for T cells to enter lymph nodes. It is expressed on lymph node endothelia, whereas the L-selectin to which it binds is on the T cell.

Conversely, under other circumstances CD34 has been shown to block mast cell, eosinophil and dendritic cell precursor adhesion, and to facilitate opening of vascular lumens (e.g., as described in Drew et al., Immunity. 22 (1): 43-57, 2005, incorporated herein by reference). CD34 may also play a more selective role in chemokine-dependent migration of eosinophils and dendritic cell precursors (e.g., as described in Blanchet et al., Blood. 110 (6): 2005-12, 2007, incorporated herein by reference).

In some embodiments, HSCs are isolated from umbilical cord blood (e.g., human umbilical cord blood). In some embodiments, HSCs isolated from umbilical cord blood are sorted (e.g., by FACS) based on the protein expression profile on their surface. In some embodiments, a HSC with surface expression of CD34 (referred to as "CD34+," "+" means there is detectable expression of the surface molecule, such as CD34) is selected (e.g., collected after sorting) for differentiation to obtain monocytes. In some embodiments, the CD34+ HSC further expresses other surface molecules such as CD90 (CD90+). In some embodiments, the HSC does not express a lineage specific marker on its surface (referred to as "lineage-," "-" means there is no detectable expression of the surface molecule, such as a lineage specific marker). In some embodiments, a HSC does not express CD235a (CD235a-). In some embodiments, a HSC does not express CD38 (CD38-). In some embodiments, a HSC does not express CD45RA (CD45RA-). In some embodiments, a HSC does not express CD235a (CD235-), CD38 (CD38-), or CD45RA (CD45RA-). In some embodiments, a HSC used for differentiation to obtain monocytes has a surface expression profile of lineage - CD235a-CD34+CD38-CD45RA-CD90+.

In some embodiments, the HSC used for differentiation to obtain monocytes is generated from pluripotent stem cells (e.g., ES cells or iPS cells). Such HSC may also be referred to as "PS-derived HSC" herein. Methods for generating HSCs from pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells) have been described in the art (e.g., in Amabile et al., Blood 121, 1255-1264, 2013; Doulatov et al., Cell Stem Cell 13, 459-470, 2013; Ledran et al., Cell Stem Cell 3, 85-98, 2008; Sturgeon et al., Nature

Biotechnology 32, 554-561, 2014, Suziki et al., Mol. Ther 21, 1423-1431, 2013, the entire contents of each of which are incorporated herein by reference). In some embodiments, HSCs differentiated from pluripotent stem cells are sorted (e.g., by FACS) prior to being

differentiated into monocytes. In some embodiments, a CD34+ PS-HSC is selected (e.g., collected after sorting) for differentiation to obtain monocytes. In some embodiments, the CD34+ PS-HSC further expresses other surface molecules such as CD45 (CD45+). In some embodiments, a PS-HSCs with a surface expression profile of CD34+CD45+ is used for differentiation of monocytes. Such CD34+CD45+ PS-HSCs possess robust progenitor activity.

In some embodiments, a CD34+ cell is differentiated into a monocyte. Herein, a "CD34+ cell" refers to a cell that has detectable CD34 expression on its surface. For example, the HSC described herein, if expression CD34+ on cell surface, may also be referred to as a CD34+ cell. Any known methods of detecting the expression of a protein on a cell surface may be used. Examples of such methods include, without limitation: western blotting, immuno staining, and fluorescence-activated cell sorting (FACS). The skilled artisan is familiar with these methods. Examples of cells that are "CD34+ cells" include, without limitation: hematopoietic cells found in the umbilical cord and bone marrow (e.g., HSC), certain mesenchymal stem cells, endothelial progenitor cells, endothelial cells of blood vessels, mast cells, a sub-population of dendritic cells in the interstitium and around the adnexa of dermis of skin, as well as cells in certain soft tissue tumors. The presence of CD34 on non- hematopoietic cells in various tissues has been linked to progenitor and adult stem cell phenotypes.

In some embodiments, the CD34+ cells of the present disclosure are differentiated from pluripotent stem cells (e.g., ES cells or an iPS cells) by culturing iPS cells or ES cells in a differentiation media (e.g., a media containing FHB-hTERT, as described in Chang et al., PLoS ONE 6(10):e25761, 2011, incorporated herein by reference). Any methods known in the art for the differentiation of pluripotent stem cells into CD34+ cells may be used in accordance with the present disclosure. CD34+ cells that also lack the expression of lineage specific markers are of an undifferentiated, primitive form; i.e., they are multipotent stem cells. In some embodiments, the CD34+ cell differentiated from a pluripotent stem cell also expresses CD45 on its surface (CD45+). In some embodiments, the CD34+ cell CD34+ cell differentiated from a pluripotent stem cell has a surface expression profile of CD34+CD45+.

In some embodiments, differentiating the cell (e.g., an iPS cell, a HSC, or a CD34+ cell) into a monocyte involves contacting the cell with an effective amount of one or more protein factors that induce the differentiation of the cell. Non-limiting examples of protein factors that may be used to induce the differentiation of the cell (e.g., the iPS cell, the HSC, or the CD34+ cell) into monocytes include, without limitation: macrophage colony- stimulating factor (M-CSF), stem cell factor (SCF), receptor-type tyro sine-protein kinase FLT3 (Flt-3), interleukin-3 (IL-3), or combinations thereof. In some embodiments, the cell is contacted with an effective amount of M-CSF. In some embodiments, the cell is contacted with an effective amount of hSCF. In some embodiments, the cell is contacted with an effective amount of M- CSF and SCF. In some embodiments, the cell is contacted with an effective amount of M-CSF and Flt-3. In some embodiments, the cell is contacted with an effective amount of M-CSF and IL-3. In some embodiments, the cell is contacted with an effective amount of SCF and Flt-3. In some embodiments, the cell is contacted with an effective amount of SCF and IL-3. In some embodiments, the cell is contacted with an effective amount of Flt-3 and IL-3. In some embodiments, the cell is contacted with an effective amount of M-CSF, SCF, and Flt-3. In some embodiments, the cell is contacted with an effective amount of M-CSF, SCF, and IL-3. In some embodiments, the cell is contacted with an effective amount of SCF, Flt-3, and IL3. In some embodiments, the cell is contacted with an effective amount of M-CSF, SCF, Flt-3 and IL-3. In some embodiments, human protein factors are used. For example, the macrophage colony- stimulating factor (M-CSF), stem cell factor (SCF), receptor-type tyrosine -protein kinase FLT3 (Flt-3), or interleukin-3 (IL-3) may be human macrophage colony-stimulating factor (M-CSF), human stem cell factor (SCF), human receptor-type tyrosine-protein kinase FLT3 (Flt-3), or human interleukin-3 (IL-3), respectively.

In some embodiments, prior to being differentiated, the cell (e.g., the iPS cell, the HSC, or the CD34+ cell) is expanded to produce a population of the cell. "Expanding" the cell (e.g., the iPS cell, the HSC, or the CD34+ cell) or "expansion" of the cell refers to the process where a homogenous population of cells are generated from a smaller number of cells. For example, in some embodiments, no more than 500, no more than 400, no more than 300, no more than 200, no more than 100, no more than 50, no more than 25, no more than 10, no more than 5, or a single cell (e.g., iPS cells, HSCs, or CD34+ cells) are expanded to generate a population of more than 10³, more than 10⁴, more than 10⁵, or more than 10⁶, or more than 10⁷ cells. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170m 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 cells (e.g., iPS cells, HSCs, or CD34+ cells) are expanded to generate a population of more than 10³, more than 10⁴, more than 10⁵, or more than 10⁶, or more than 10⁷ cells. In some embodiments, no more than 5 (e.g., 1, 2, 3, 4, 5) cells are used to generate a population of more than 1000, more than 2000, more than 3000, more than 4000, more than 5000, more than 6000, more than 7000, more than 8000, more than 9000, or more than 10000 cells. In some embodiments, no more than 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) cells (e.g., iPS cells, HSCs, or CD34+ cells) are expanded to generate a population of more than 1000, more than 2000, more than 3000, more than 4000, more than 5000, more than 6000, more than 7000, more than 8000, more than 9000, or more than 10000 cells.

The population of cells (e.g., iPS cells, HSCs, or CD34+ cells) generated by expanding a smaller number of cells is homogeneous. Being homogeneous means that at least 50% of the cells in the population maintain the same biological property (e.g., expression of any one of the cells surface markers as described herein) and functionality (e.g., pluripotency or

multipotency). It is to be understood that homogeneity does not require all the cells to express the same set of markers. For example, cells that express any one of the HSC surface markers and maintain the functionalities of HSC can be considered a homogenous population of HSCs. For example, a population of HSCs (e.g., more than 10⁵ HSCs) expanded from a smaller number of HSCs (no more than 25 HSCs) contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of cells that maintain the same biological property (e.g., CD34+CD38-) and functionality (e.g., hematopoietic functionality) as the HSCs that are used for the expansion. In some embodiments, a population of HSCs (e.g., more than 10⁵ HSCs) expanded from a smaller number of HSCs (no more than 25 HSCs) contains 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of cells that maintain the same biological property (e.g., CD34+CD38-) and functionality (e.g., hematopoietic functionality) as the HSCs that are used for the expansion.

In some embodiments, a population of iPS cells (e.g., more than 10⁵ iPS cells) expanded from a smaller number of iPS cells (no more than 25 iPS cells) contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of cells that maintain the same biological property (e.g., expression of pluripotent stem cell markers) and functionality (e.g.,

pluripotency) as the iPS cells that are used for the expansion. In some embodiments, a population of iPS cells (e.g., more than 10⁵ iPS cells) expanded from a smaller number of iPS cells (no more than 25 iPS cells) contains 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of cells that maintain the same biological property (e.g., expression of pluripotent stem cell markers) and functionality (e.g., pluripotency) as the HSCs that are used for the expansion.

In some embodiments, a population of CD34+ cells (e.g., more than 105 CD34+ cells) expanded from a smaller number of CD34+ cells (no more than 25 CD34+ cells) contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of cells that maintain the same biological property (e.g., CD34+) and functionality (e.g., multipotency) as the CD34+ cells that are used for the expansion. In some embodiments, a population of CD34+ cells (e.g., more than 105 CD34+ cells) expanded from a smaller number of CD34+ cells (no more than 25 CD34+ cells) contains 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of cells that maintain the same biological property (e.g., CD34+) and functionality (e.g., multipotency) as the HSCs that are used for the expansion.

Methods of expanding cells (e.g. HSCs) are known in the art, e.g., as described in PCT Application Publication WO2016210292 and PCT Application PCT/US2017/022534, incorporated herein by reference). The methods exploit agents (e.g., small molecules) that modulate histone methylation, inhibit TGFP signaling; inhibit p38 signaling, activate canonical Wnt signaling, modulate histone acetylation, and/or activate a ubiquitin ligase complex. As described in WO2016210292 and PCT/US2017/022534, a variety of agents (e.g., small molecules) may be used to achieve the desired biological activity. For example, for histone methylation modulation, a histone demethylase inhibitor may be used. In some embodiments, the histone demethylase inhibitor is selected from the group consisting of LSDl inhibitor IV RN-1, LSDl inhibitor II S2101, LSDl inhibitor LSD1-C76, LSDl inhibitor III CBB 1007, LSDl inhibitor I, and Tranylcypromine. In some embodiments, for inhibition of TGFP signaling, an agent that inhibits a protein that propagates TGFP signaling may be used. In some

embodiments, the agent that inhibits a protein that propagates TGFP signaling is selected from the group consisting of ALK5 inhibitor II, LY364947, A83-01, and DMH1. In some embodiments, for inhibition of p38 signaling, an agent that inhibits a protein that propagates p38 signaling is used. In some embodiments, the agent that inhibits a protein that propagates p38 signaling is SB203580. In some embodiments, an agent that activate canonical Wnt signaling is an agent that inhibits a protein that promotes β-catenin degradation. In some embodiments, the agent that inhibits a protein that promotes β-catenin degradation is selected from the group consisting of CHIR99021, lithium chloride, BIO, and FGF2. In some embodiments, the agent that modulates histone acetylation is an agent that inhibits a histone deacetylase. In some embodiments, the agent that inhibits a histone deacetylase is selected from the group consisting of Trichostatin A, valproic acid, butyrylhydroxamic acid, and istodax. In some embodiments, the agent that activates a ubiquitin ligase complex is a cereblon. In some embodiments, the cereblon is selected from the group consisting of pomalidomide, lenalidomide, and thalidomide. In some embodiments, an agent that inhibits aryl hydrocarbon receptor signaling (such as SRI or an analog thereof) is also used. In some embodiments, the agent that inhibits aryl hydrocarbon receptor signaling (such as SRI or an analog thereof) is UM171, or structural analogs thereof.

In some embodiments, the methods of expanding the cell (e.g., the iPS cell, the HSC, or the CD34+ cell) used in accordance with the present disclosure comprises contacting the cell with an effective amount of A83-01, pomalidomide, and UM171. In some embodiments, during the expansion, the cell is further contacted with an effective amount of one or more of SCF, thrombopoietin (TPO), IL3, and Flt3L. In some embodiments, the cell is contacted with an effective amount of SCF, thrombopoietin (TPO), IL3, and Flt3L. In some embodiments, the methods of expanding the cell (e.g., the iPS cell, the HSC, or the CD34+ cell) comprises culturing the cell in an expansion media. In some embodiments, the expansion media comprises an effective amount of SCF (e.g., human SCF), TPO (e.g., human TPO), IL-3 (e.g., human IL-3), Flt3L (e.g., human Flt3L), A83-01, pomalidomide, and UM171. In some embodiments, the expansion media comprises SCF (e.g., human SCF) at 100 ng/ml, TPO (e.g., human TPO) at 100 ng/ml, IL-3 (e.g., human IL-3) at 50 ng/ml, Flt3L (e.g., human Flt3L) at 50 ng/ml, A83-01 at 1 μΜ, pomalidomide at 2 μΜ, and UM171 at 35 nM.

Expansion of the cell (e.g., the iPS cell, the HSC, or the CD34+ cell) maintains the biological properties or functionality of the cell. For example, the population of the cell produced by expanding a small number of the cell may have a less than 20% variation in the surface expression profile, either in the number of surface proteins or in their respective expression level. In some embodiments, the population of the cell produced by expanding a small number of the cell may have a less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, or no variation in the surface expression profile, either in the number of surface proteins or in their respective expression level. In some embodiments, the population of the cell produced by expanding a small number of the cell may have a 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or no variation in the surface expression profile, either in the number of surface proteins or in their respective expression level. In some embodiments, a population of HSCs produced by expanding a small number (e.g., no more than 100, no more than 25, or no more than 5) of HSCs using the methods described herein have a surface expression profile of lineage- CD235a-CD34+CD38-CD45RA-CD90+. In some embodiments, a population of CD34+ cells produced by expanding a small number (e.g., no more than 100, no more than 25, or no more than 5) of CD34+ cells using the methods described herein have a surface expression profile of CD34+CD45+. In some embodiments, the population of cells produced by expanding the cell using the methods described herein maintains the pluripotency (e.g., for iPS cells) or multipotency (e.g., for HSC) of the cell before expansion.

The cell (e.g., the iPS cell, the HSC, or the CD34+ cell) of the present disclosure may be subjected to differentiation either directly or after expansion. Expansion of the cell enables the use of a small number of cells to generate a large number of monocytes, and ultimately microglia-like cells. Monocytes differentiated from the cell (e.g., the iPS cell, the HSC, or the CD34+ cell) using the methods described herein express any one of CD14, CD16, CDl lb, and/or CD33. For example, in some embodiments, the monocyte expresses CD 14 on its surface. In some embodiments, the monocyte expresses CD16 on its surface. In some embodiments, the monocyte expresses CDl lb on its surface. In some embodiments, the monocyte expresses CD33 on its surface. In some embodiments, the monocyte expresses CD14 and CD16 on its surface. In some embodiments, the monocyte expresses CD14 and CDl lb on its surface. In some embodiments, the monocyte expresses CD14 and CD33 on its surface. In some embodiments, the monocyte expresses CD16 and CDl lb on its surface. In some embodiments, the monocyte expresses CD16 and CD33 on its surface. In some embodiments, the monocyte expresses CDl lb and CD33 on its surface. In some

embodiments, the monocyte expresses CD14, CD16, and CDl lb on its surface. In some embodiments, the monocyte expresses CD14, CD16, and CD33 on its surface. In some embodiments, the monocyte expresses CD16, CDl lb, and CD33 on its surface. In some embodiments, the monocyte expresses CD14, CD16, CDl lb, and CD33 on its surface. It is to be understood that not all monocytes differentiated using the methods described herein express the same surface markers described herein.

In some embodiments, the expression level of any one of CD14, CD16, CDl lb, and CD33 increases as the cell (e.g., the iPS cell, the HSC, or the CD34+ cell) is differentiated into a monocyte, compared to the expression level of these markers prior to differentiation. For example, compared to an undifferentiated cell (e.g., the iPS cell, the HSC, or the CD34+ cell), the expression level of any one of CD14, CD16, CD1 lb, and CD33 on a monocyte may be increased by at least 30%, at least 40%, least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000- fold, or more. In some embodiments, compared to an undifferentiated cell (e.g., the iPS cell, the HSC, or the CD34+ cell), the expression level of any one of CD14, CD16, CD1 lb, and CD33 on a monocyte may be increased by 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2- fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more.

In some embodiments, the monocyte dose not express or has decreased expression (e.g., decreased by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%) of CD34 and/or CD45 on its surface, compared to the cell (e.g., the iPS cell, the HSC, or the CD34+ cell) before differentiation.

In some embodiments, when the population of the cell produced by expanding the cell are used for differentiation, about 20-100% of the population of the cell are differentiated into monocytes. For example, about 20%- 100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%- 50%, 20%-40%, 20%-30%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%- 50%, 30%-40%, 40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%- 100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%- 70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the population of the cell are differentiated into monocytes. In some embodiments, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the population of the cell are differentiated into monocytes. In some embodiments, at least 20% of the population of the cell are differentiated into monocytes. For example, at least 20%, least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the population of the cell may be differentiated into monocytes. In some embodiments, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the population of the cell are differentiated into monocytes.

The monocytes are further subjected to differentiation to generate microglia-like cells. Accordingly, some aspects of the present disclosure provide methods of differentiating a monocyte into a microglia-like cell. In some embodiments, the method of differentiating a monocyte into a microglia-like cell comprises contacting the monocyte with an effective amount of granulocyte macrophage colony- stimulating factor (GM-CSF) and/or interleukin-34 (IL-34). In some embodiments, the monocyte is contacted with an effective amount of GM- CSF. In some embodiments, the monocyte is contacted with an effective amount of IL-34. In some embodiments, the monocyte is contacted with an effective amount of GM-CSF and IL- 34. In some embodiments, the GM-CSF or IL-34 may be human GM-CSF or human IL-34, respectively.

In some embodiments, about 20-100% of the monocytes are differentiated into microglia-like cells. For example, about 20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%- 60%, 20%-50%, 20%-40%, 20%-30%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%- 60%, 30%-50%, 30%-40%, 40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%- 50%, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%- 80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the monocytes are differentiated into microglia-like cells. In some embodiments, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of monocytes are differentiated into microglia-like cells. In some embodiments, at least 20% of the monocytes are differentiated into microglia- like cells. For example, at least 20%, least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the monocytes may be differentiated into microglia-like cells. In some embodiments, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the monocytes are differentiated into microglia-like cells.

In some embodiments, the microglia-like cells obtained using the methods described herein have increased CX3CR1/CCR2 expression ratio. "CX3CR1/CCR2 expression ratio" refers to the relative expression level of two cell surface makers CXCR1 and CCR2, and is calculated by dividing the expression level of CX3CR1 by the expression level of CCR2. Methods of measuring the expression levels of the two surface markers are known to those skilled in the art, e.g., western blotting, immuno staining, and/or FACS. Microglia-like cells distinguish from monocytes in that the CX3CR1/CCR2 expression ration increases, e.g., as described in Ohgidani et al., Scientific reports 4:4957, 2014, incorporated herein by reference. Monocytes express low levels of CX3CR1 and high levels of CCR2, while microglia-like cells express low levels of CCR2 and high levels of CX3CR1. In some embodiments, the

CX3CR1/CCR2 expression ratio on a microglia-like cell is increased by at least 30% compared to a monocyte. For example, the CX3CR1/CCR2 expression ratio on a microglia-like cell may be increased by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more. In some embodiments, the CX3CR1/CCR2 expression ratio on a microglia-like cell is increased by 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more. In some embodiments, the CX3CR1/CCR2 expression ratio on a microglia-like cell is increased by at least 50%.

Other aspects of the present disclosure provide microglia-like cells generated using the methods described herein and their characterization. The microglia-like cell generated using the methods described adopt a similar ramified morphology. The microglia-like cell generated using the methods described herein have comparable activity biological activities and functionalities as a natural microglia (e.g., resident microglia). Having "comparable activity" means that the microglia-like cells retains at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, 100%, or higher) of any biological activity or functionality that a natural microglia has. For example, the microglia-like cell generated using the methods described herein secrets cytokines upon stimulation. Microglia is known to have different cytokine secretion profiles in activated (upon stimulation, e.g., by a pathogen) or inactive (no stimulation, e.g., by a pathogen) states. For example, the microglia-like cell generated using the methods described herein secretes cytokines upon stimulation (e.g., using INF-γ and/or LPS). In some embodiments, compared to an unstimulated microglia-like cell, the stimulated microglia-like cell secretes a higher level of a cytokine, e.g., a cytokine that plays a role in mediating neuroinflammation and/or microglia activation. In some embodiments, the cytokine is selected from the group consisting of CCL2, MIP-la/b, CXCL10, IL-6, TNF-a, Serpin El, and combinations thereof. In some embodiments, the expression level of any one of CXCL10, CCL2, IL-6, or MIP-la/b is at least 2-10 times higher (e.g., 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2- 3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times higher) than the respective expression level in an unstimulated microglia-like cell. In some embodiments, the expression level of CCL2 in the microglia-like cell is 2-3 times higher upon stimulation, compared to before stimulation. In some embodiments, the expression level of MIP-la/b in the microglia-like cell is 2-3 times higher upon stimulation, compared to before stimulation. In some embodiments, the expression level of CXCLIO in the microglia-like cell is 5-6 times higher upon stimulation, compared to before stimulation. In some embodiments, the expression level of IL-6 in the microglia-like cell is 3-4 times higher upon stimulation, compared to before stimulation. In some embodiments, the expression level of Sepin El in the microglia-like cell is 1-2 times higher upon stimulation, compared to before stimulation. In some embodiments, the expression level of TNF-a in the microglia-like cell is 1-2 times higher upon stimulation, compared to before stimulation. In some embodiments, a microglia-like cell generated directed from a CD34+ cell without expansion has an increased expression of a cytokine selected from the group consisting of CCL1, CCL2, MlPla/b, CC15, CXCL1, CXCLIO, GM-CSF, IFN-γ, IL- RA, IL-6, IL-8, SerpinEl, and TNF-a upon stimulation, compared to before stimulation.

In some embodiments, microglia-like cell generated using the methods described herein is phagocytic. Being "phagocytic" means the microglia-like cell has the ability to engulf and destroy pathogens, waste material, and other particulate matter, similar to such ability of phagocytes and macrophages. In some embodiments, the phagocytic microglia-like cell expresses Allograft inflammatory factor 1 (IBA1) on its surface. "IBA1" is a protein that is specifically expressed by macrophages and microglia. In some embodiments, at least 50% of the microglia-like cells generated using the methods described herein have surface IBA1 expression. For example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% of the microglia-like cells generated using the methods described herein have surface IBA1 expression. In some embodiments, 95-100% of the microglia-like cells generated using the methods described herein have surface IBA1 expression.

The methods described herein are advantageous compared to existing methods (e.g., as described in Ohgidani et al., 2014, and Muffat et al., 2016), at least because the microglia-like cells generated using the methods described herein are homogenous (i.e., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%) of the cells are similar in their morphology and activity. Starting the differentiation from a pluripotent cell (e.g., iPS) or a multipotent cell (e.g., HSC) gives more robust results (e.g., as compared to the methods of Ohgidani), which may partially be attributed to the fact that the pluripotent cell or multipotent cell has not committed to a specific lineage (in contrast to using committed monocytes for differentiation in Ohgidani).

While the microglia-like cell generated using the methods described herein has comparable morphology, biological activities, and functionalities as a natural microglia, it may have different protein expression profiles, cytokine secretion profiles, and/or epigenetic profiles, compared to a natural microglia (e.g. resident microglia). The microglia-like cells generated using the methods described herein may be used in platforms to model microglia- related diseases, to study various functionalities of microglia, and for the identification of therapeutic targets and drugs for treating microglia-related diseases.

Agents and effective amount

Different agents (e.g., protein factors or small molecules) are used in the methods of generating microglia-like cells described herein. The cells used for differentiation (e.g., iPS cells, HSC, CD34+ cells) are contacted with an effective amount of one or more appropriate agents to induce differentiation of the cell (e.g., the iPS cell, the HSC, or the CD34+ cell) into a monocyte or to induce differentiation of the monocyte into a microglia-like cell. When a protein factor is used, in some embodiments, the protein factor is a human protein factor.

An "effective amount," as used herein refers to an amount sufficient to induce differentiation of a cell when the cell is contacted with the agent. The effective amount for each agent (e.g., protein factor or small molecule) varies. For the sole purpose of illustration, the amount of each agent that may be used in accordance with the present disclosure is exemplified in Table 1. It is to be understood that the concentrations of the agent listed in Table 1 is not considered to be limiting. One skilled in the art is able to determine and adjust the effective amount for each agent (e.g., protein factor or small molecule).

"Contacting" means the cell is physically in close proximity with the agent such that it is considered to "touch" the cell. As such, supplementing a differentiation media or an expansion media with the appropriate agents for inducing differentiation or expansion and culturing the cell in such supplemented media is considered "contacting" the cell with the agents.

In some embodiments, the methods described herein involves culturing the cell to be differentiated into microglia-like cells in a media that induces differentiation (referred to as "differentiation media"), and optionally in a media that induces expansion (referred to as "expansion media"). Two different types of differentiation medium are used for the two steps of differentiation (e.g., from iPS or HSC or CD34 cells to monocytes and from monocytes to microglia-like cells, referred to as the "first differentiation media" and the "second

differentiation media," respectively). In some embodiments, the cell (e.g., iPS, HSC, or CD34+ cell) is cultured in the first differentiation media for a period of time to allow the cell to differentiate into a monocyte, followed by culturing in the second differentiation media for a period of time to allow the monocyte to differentiate into a microglia-like cell. In some embodiments, prior to being cultured in the first differentiation media, the cell is cultured in the expansion media for a period of time to produce a population of the cell. The effective components of each media and their respective concentrations are listed in Table 1. In some embodiments, the media may further be supplemented by other agents, such as those used in standard cell culture conditions (e.g., Penicillin/Streptomycin). One skilled in the art is able to determine each of these additional agents to use and their respective concentrations.

In some embodiments, the "contacting" may be carried out for a period of time (e.g., no more than 14 days) for each step (e.g., expansion, differentiation to obtain monocytes, or differentiation to obtain microglia-like cells). For example, for expansion, the cell is cultured in the expansion media for no more than 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). In some embodiments, the cell is cultured in the first differentiation media for no more than 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). In some embodiments, the cell is cultured in the second differentiation media for no more than 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). As such, the methods described herein, without an expansion step, generates microglia-like cells in no more than 28 days; and with an expansion step, generates microglia-like cells in no more than 42 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days). The methods requires non-laborious steps such as culturing the cells and are thus advantageous over the methods described in Muffat et al., which requires laborious steps and takes over 72 days.

In some embodiments, the method of generating a microglia-like cell described herein is carried out in vitro. In some embodiments, the method of generating a microglia-like cell described herein is carried out ex vivo. In some embodiments, the cell (e.g., iPS, HSC, or CD34 cell) is from a mammal. In some embodiments, the mammal is a rodent, such as a mouse or a rat. In some embodiments, the mammal is a primate, such as a human.

Table 1. Agents for Cell Expansion and/or Differentiation

MEE VS E YC S HMIGS GHLQS LQR

LIDSQMETSCQITFEFVDQEQLK

macrophage

First 5-100 DPVCYLKKAFLLVQDIMEDTMR colony- different ng/ml Peprotech FRDNTPN AIAIVQLQELS LRLKS stimulating

iation (e.g., 30 CFTKDYEEHDKACVRTFYETPL factor (M- media ng/ml) QLLEKVKNVFNETKNLLDKDW CSF)

NIFS KNCNNS F AEC S S QGHERQS EGS

MEGICRNRVTNNVKDVTKLVA

First

NLPKD YMITLKY VPGMD VLPS H

different Peprotech

50-300 CWISEMVVQLSDSLTDLLDKFS iation

Stem cell ng/ml NISEGLSNYSIIDKLVNIVDDLVE media

factor (SCF) (e.g., 100 C VKENS S KDLKKS FKS PEPRLFT and

ng/ml) PEEFFRIFNRS ID AFKDFV V AS ET expansio

SDCVVSSTLSPEKDSRVSVTKPF

n media

MLPPVA

TQDCSFQHSPISSDFAVKIRELSD YLLQDYPVTVASNLQDEELCGG

receptor-type First 5-100

LWRLVLAQRWMERLKTVAGSK

tyrosine- different ng/ml Peprotech

MQGLLERVNTEIHFVTKCAFQP

protein kinase iation (e.g., 50

PPS CLRF VQTNIS RLLQETS EQL FLT3 (Flt-3) media ng/ml)

VALKPWITRQNFSRCLELQCQP DSSTLPPPWSPRPLEATAPTA

First

APMTQTTSLKTSWVNCSNMIDE

different 5-100

IITHLKQPPLPLLDFNNLNGEDQ

iation ng/ml

interleukin-3 DILMENNLRRPNLEAFNRAVKS media (e.g., 30 Peprotech

(IL-3) LQNASAIESILKNLLPCLPLATA and ng/ml or

APTRHPmiKDGDWNEFRRKLTF expansio 50 ng/ml)

YLKTLEN AQ AQQTTLS LAIF

n media

granulocyte Second 1-100 M AP ARS PS PS TQPWEH VN AIQE macrophage different ng/ml ARRLLNLS RDT A AEMNET VE VI

Peprotech

colony- iation (e.g., 10 SEMFDLQEPTCLQTRLELYKQG stimulating media ng/ml) LRGS LTKLKGPLTMM AS H YKQ factor (GM- HCPPTPETSCATQIITFESFKENL CSF) KDFLLVIPFDCWEPVQE

NEPLEMWPLT QNEECTVTGFLRDKLQYRSRLQ YMKHYFPINYKISVPYEGVFRIA N VTRLQRAQ VS ERELRYLW VL

Second 10-500

interleukin- VSLSATESVQDVLLEGHPSWKY different ng/ml

34 (IL-34) Peprotech LQE VETLLLN VQQGLTD VE VS P iation (e.g., 100

KVES VLS LLN APGPNLKLVRPK

media ng/ml)

ALLDNCFRVMELLYCSCCKQSS VLNWQDCEVPSPQSCSPEPSLQ YAATQLYPPPPWSPSSPPHSTGS VRPVRAQGEGLLPHHHHHHHH

S P APP ACDLRVLS KLLRDS H VL HS RLS QCPE VHPLPTP VLLP A VD

50-300 FSLGEWKTQMEETKAQDILGAV

Expansi

Thrombopoie ng/ml TLLLEGVM AARGQLGPTCLS S L on Peprotech

tin (TPO) (e.g., 100 LGQLS GQVRLLLGALQS LLGTQ media

ng/ml) LPPQGRTT AHKDPN AIFLS FQHL

LRGKVRFLMLVGGSTLCVRRAP PTT A VPS RTS LVLTLNEL

TQDCSFQHSPISSDFAVKIRELSD YLLQDYPVTVASNLQDEELCGG

FMS-like 5-100

Expansi LWRLVLAQRWMERLKTVAGSK tyrosine ng/ml

on Peprotech MQGLLERVNTEIHFVTKCAFQP kinase 3 (e.g., 30

media PPS CLRF VQTNIS RLLQETS EQL ligand (Flt3L) ng/ml)

VALKPWITRQNFSRCLELQCQP DSSTLPPPWSPRPLEATAPTA

Expansi 0.1-10

A83-01 on μΜ (e.g., Stemgent Inc. NA

media 1 μΜ)

Pomalidomid Expansi 0.1-10

Celgene NA

e on μΜ (e.g., media 2μΜ)

Expansi 10-100

UM171 on nM (e.g., ApexBio NA media 35 nM)

Table 2. Cell Surface Makers

CD l ib Positive on Monocyte NP_001139280.1

NP_000623.2

CD33 Positive on Monocyte NP_001171079.1

NP_001076087.1

NP_001763.3

IBA1 Positive on Microglia-like NP_001305899.1

cell NP_001614.3

NP_116573.1

CD235a Negative on HSC NP_002090.4

NP_001295116.1

NP_001295119.1

CD38 Negative on HSC NP_001766.2

CD45RA Negative on HSC NP_001254727.1

NP_002829.3

NP_563578.2

Some of the embodiments, advantages, features, and uses of the technology disclosed herein will be more fully understood from the Examples below. The Examples are intended to illustrate some of the benefits of the present disclosure and to describe particular embodiments, but are not intended to exemplify the full scope of the disclosure and, accordingly, do not limit the scope of the disclosure.

EXAMPLES

Example 1: Induced microglia (iMicroglia) from hematopoietic and pluripotent stem cells.

As the resident immune cells of the central nervous system, microglia have been shown to play crucial roles in surveillance, pruning/neuromodulation, and inflammation, among other functions in both the healthy and diseased brain. In their resting state, microglia actively survey their environment and when an insult is detected, they enter an activated state to carry out their immune response. Normally, these cells respond to neuronal damage by changing their morphology, proliferating, and removing damaged cells from the affected environment. They can also secrete cytokines and chemokines, prostaglandins, and NO and reactive oxygen species, in order to increase and direct the immune response. Microglia are thought to contribute to diseases such as, but not limited to, Alzheimer's disease, Parkinson's disease, multiple sclerosis, schizophrenia, prion disease, and dementia induced by ageing. Though it is understood that microglia can have harmful effects in many neurological diseases, specific mechanisms are not yet well understood, so it is important to have a robust platform in order to study microglia and its role in disease. Microglia are related to aging, schizophrenia,

Alzheimer's, and autism, but have not been previously used in cellular models of mental disorders. Microglia may serve as vehicles for gene modifications to the nervous system.

In order to study microglia in vitro, cells must be isolated from the post mortem brain of a patient or an animal model. However, it would be beneficial to test and monitor the state of the microglia while the patient or subject is still alive, which is not possible as microglia must be isolated from the brain and would require invasive procedures. Peripheral blood however, is easily isolated from a living subject. Monocytes in the peripheral blood are known to be recruited into the adult brain, especially under inflammatory conditions. By

differentiating cells in the peripheral blood into microglia like cells in vitro, a platform to study can be developed in order to study interventions or characteristics of microglia in various disease states. With a platform to perform small molecule screening, a variety of different drugs and chemical compounds can be tested on microglia to identify therapeutic targets as well as drugs effective in treating microglial dysfunction.

Currently, there are only a handful of protocols established to induce microglia- like cells from cells contained in the peripheral blood. One protocol derives microglia-like cells from induced pluripotent stem (iPS) cells and embryonic stem (ES) cells (Muffat et al., 2016). This protocol however is very laborious and takes over 74 days. There is also a protocol to differentiate monocytes into microglia-like cells. However, this protocol requires a very large amount of cells (Ohgidani et al., 2014), which limits its scalability. In order to circumvent these issues, a protocol to derive a robust population of microglia-like cells from hematopoietic progenitors and stem cells, as well as purified hematopoietic stem cells and induced pluripotent stem cells was developed herein. This protocol can be done with a starting number of 1, 10, 100 or more cells, thus expanding its applicability for clonal derivation of gene edited cells or patient cells for disease modeling. This work could lead to the generation of a platform to model healthy and diseased states of microglia, with which to study various functions and interventions. In addition, a variety of different drugs and chemical compounds can be tested on these microglia-like cells to identify therapeutic targets as well as drugs effective in treating microglial dysfunction.

METHODS Cell sorting

CD34 enriched human cord blood were thawed according to the provider's instructions and plate sorted as single cells or cell numbers indicated in each experiment using a FACS Aria (BD Biosciences). Cells were sorted into the central 60 wells of a 96 well round bottom plate (Falcon) with 100 μΐ of media per well. The wells in the perimeter of the plate were filled with 100 μΐ of lx PBS. Cells were stained with the listed antibodies below at a 1: 100 dilution in sample media (FBS, 0.5M EDTA, PBS and P/S) for 1 hour on ice. After staining, the cells were washed and re-suspended in sample media at 1:2000 propidium iodide solution (Sigma).

Immunophenotypic HSCs (Lineage-CD34+CD38-CD45RA-CD90+, Fig. 3A) or CD34+ HSPCs (Lineage-CD34+, Fig. 3B) were directly sorted into the wells. Plated cells were cultured in at 37°C, 5% C02, 5% 02 in a humidified incubator. Media was changed every 3 days. The first media change was accomplished by adding 100 μΐ of media per well.

Subsequent media changes were accomplished by removing ΙΟΟμΙ of media and adding ΙΟΟμΙ freshly prepared complete media per well.

HSC and CD34+ cell staining antibodies: APC anti-human Lineage (CD3/14/19/20/56, BioLegend), APC anti-human CD235a (Clone HIR2, eBioscience), Brilliant Violet 421 anti- human CD34 (Clone 561, BioLegend), APC/Cy7 anti-human CD38 (Clone HIT2, BioLegend), PerCP/Cy5.5 anti-human CD45RA (Clone HI100, BioLegend), PE/Cy7 antihuman CD90 (Clone 5E10, BioLegend), PE anti-human CD49F (Clone eBioGoH3, eBioscience). Gating strategy used is shown in Figures 3A-3D.

Expansion of HSCs and CD34+ cells in culture

Following plate sorting, cells were cultured in expansion media containing StemSpan SFEM II (StemCell Technologies) with lx Penicillin/Streptomycin (P/S, 10,000 U/ml, Gibco), recombinant human SCF (Peprotech), recombinant human TPO (Peprotech), recombinant human Flt-3 (Peprotech), recombinant human IL-3 (Peprotech) and supplemented with

StemRegeninl (SRI, ApexBio), or UM171 (ApexBio), and DMSO (Sigma): SRI, UM171, expansion media without UM171, expansion media + SRI, expansion and DMSO (control). Cells remained in culture at 37°C, 5% C0₂, 5% 0₂ for a total of 14 days and media was changed every 2 to 3 days throughout the duration of this protocol by removing half of the existing volume and replacing it with fresh media. On day 7 and 14, 3 wells from each condition were selected at random for flow cytometry analysis (Figure 1).

Microglia differentiation following expansion Ex vivo differentiation was either preceded by expansion or not. Differentiation was initiated from either HSCs or CD34+ cells, from this point onward, cells that began

differentiation as CD34+ cells will be referred to as "sCD34 cells" and those that began as HSCs will be referred to as "sHSCs".

I. Differentiation following expansion

Following 14 days of expansion, the expansion medias were removed and replaced with monocyte differentiation media (referred to as MonoMedia or first differentiation media from here onward), IMDM (Gibco) + 20% FBS (VWR) and lx P/S (10,000 U/ml, Gibco) supplemented with 30ng/ml recombinant human M-CSF (Peprotech), 25ng/ml recombinant human hSCF (Peprotech), 30ng/ml recombinant human Flt-3 (Peprotech) and 30ng/ml recombinant human IL-3 (Peprotech) to begin differentiation into monocytes. Cells remained in culture at 37°C, 5% C02, 5% 02 for an additional 14 days and media was changed every 2 to 3 days throughout the duration of this protocol by removing half of the existing volume and replacing it with fresh media. On day 21 and 28, 3 random wells from each condition were selected for flow cytometry analysis. Following 14 days of differentiation into monocytes, the MonoMedia was and replaced with microglia differentiation media (referred to as MicroMedia from here onward) RMPI (Corning) and lx P/S (10,000 U/ml, Gibco) supplemented with lOng/ml recombinant human GM-CSF (Peprotech) and lOOng/ml recombinant human IL-34 (Peprotech). Cells remained in culture at 37°C, 5% C02, 5% 02 for an additional 14 days and media was changed every 2 to 3 days throughout the duration of this protocol by removing half of the existing volume and replacing it with fresh media. On day 35, 2 random wells from each condition were selected for flow cytometry analysis. On day 42, the final day of this differentiation protocol, all remaining wells were analyzed by flow cytometry, phagocytosis assay, cytokine assay and immunocytochemistry (Fig 1).

II. Direct differentiation (not preceded by expansion)

CD34+ enriched human cord blood cells (AllCells) were thawed and plated into a 48 well flat bottom plate (Falcon) at a density of 50,000 cells per well in 300 μΐ of MonoMedia (Fig 1). The same differentiation protocol from the previous section was followed. .

Flow cytometry (FACS) analysis

Flow cytometry was performed using a FACS Aria and FACS Diva software. Data from flow cytometry were analyzed using Flow Jo software. For HSC and CD34+ phenotyping, antibodies Lin (APC), CD235a (APC), CD34 (BV421), CD38 (APC cy7), CD45RA (Percp cy5.5), CD90 (PEcy7) and CD49F (PE) were used. For monocytes phenotyping, antibodies CD14 (APC), CD16 (Pacific Orange/Biotin), CD33 (PE), CDl lb (PEcy7), CD34 (AF488) and CD45 (APC cy7) were used and following primary staining, cells were washed with sample media (FBS, 0.5 M EDTA, PBS and P/S) and stained with secondary antibody Streptavidin Pacific Orange. For microglia phenotyping, antibodies CCR2 (anti-mouse FITC), and

CX3CR1 (anti-goat APC) were used and following primary staining, cells were washed with sample media and stained with IgGl rat anti-mouse and Dylight649 donkey anti-goat. All primary antibodies were diluted 1: 100 and secondary antibodies were diluted 1:200 with sample media.

Cell morphology

Throughout the course of the expansion and differentiation protocol, cell

morphology was observed and documented using the Nikon Eclipse TS 100 and SPOT

RT3.

Induced microglia characterization

Following the 42-day differentiation protocol, various characterization approaches were taken to determine the functionality of the microglia like cells produced:

/. Immunocytochemistry of induced microglia

Cells were fixed by paraformaldehyde PFA 4% for 20 minutes at room temperature and quickly washed in lx PBS. Permeabilization was performed in 0.1-0.2% TRITON X, 1% bovine serum in PBS for 15 minutes at room temperature. After permeabilization, cells were washed 3 times at 5 minutes each, followed by blocking with 1% bovine serum and 0.05% Tween for 5 minutes. Primary antibody IBA-1 was incubated at 1:500 overnight at 4°C. Cells were washed 3 times with PBS and Tween 0.05% for 5 minutes, then incubated with secondary Alexa 568 donkey anti-goat (ImmunoResearch) at 1:400 for 1 hour at room temperature. After secondary antibody incubation, cells were washed once with PBS and Tween 0.05%. The cells were then incubated with DAPI (1: 1000) for 5 minutes. Following incubation, cells were washed 5 times for 5 minutes at room temperature in PBS and Tween 0.05%. Cells were then observed using Nikon Eclipse Ti.

//. Cytokine assay of induced microglia Following the differentiation protocol, cells were stimulated with 20ng/ml of

IFNgamma (Peprotech) and lOOng/ml of LPS (InvivoGen). 400μ1 of fresh media supplemented with IFN-gamma and LPS was added to each of the wells in the 48 well flat bottom plate to be tested. 400 μΐ of supernatant from the stimulated wells and 400 μΐ from the control wells which were not stimulated with IFN-gamma and LPS were added to the cytokine antibody panel membrane, following the manufacturer's instructions (R&D Systems, ARY005B). Membranes were developed and visualized for cytokine expression levels. Fold changes of expression were then analyzed in ImageJ software (NIH).

///. Phagocytosis assay of induced microglia

Phagocytic capabilities of the induced microglia-like cells were determined by fluorescent microscopy following the Phagocytosis Assay Kit (Cayman Chemical) according to the manufacturer's protocols. Following the differentiation protocol, the induced microglia- like cells were treated at 1:300 with latex beads-rabbit IgG-FITC solution in each well of the 48 well flat bottom plate. These cells were incubated for 24 hours in the same culture conditions above. The supernatant was discarded and surface bound fluorescence was removed by washing twice with RPMI (Corning). Each well was analyzed and imaged with a fluorescence microscope (Nikon Eclipse Ti).

IV. Molecular profile of induced microglia-like cells

To further investigate the molecular signature of the microglia-like cells induced from HSPCs (CB CD34+ cells) and human iPSC-derived CD34+ cells (iPSC 1 and iPSC 2) compared to their parental cell and to primary human macrophages and microglia, the molecular profile of cells coming from each of the different sources was assessed.

Differentiated cells clustered together with the primary microglia, while the macrophages were independently clustered, gathering nearer to the HSPCs (Figures 11 A and 1 IB). This data suggests that the transcription profile of induced microglia-like cells are closer to primary microglia than to their parental cells, macrophages, and/or monocytes.

Re-plating

Following the differentiation protocol, microglia-like cells were detached from the plates by treating the cells with TrypLe. Cells were replated in an untreated, 0.1% gelatin treated and fibronectin treated plate with media. RESULTS

HSC expansion

In order to find an optimum media to expand the inherently rare HSCs and CD34+ cells, including from a single cell, different combinations of existing compounds known to induce expansion of HSCs were tested. A pyrimidoindole derivative called UM171 and an aryl hydrocarbon receptor antagonist called SRI are both compounds that have been shown to promote the expansion of HSCs and CD34+ cells in vitro (Boitano, 2010; Fares, 2014). Herein, an expansion media was used that has promising results to also promote the expansion of HSCs and CD34+ cells. The expansion media used herein contains UM171. Here we tested the expansion media containing UM171, or with UM171 replaced by SRI, or UM171 or SRI alone and in combinations with one another to determine which combination would yield the most robust HSC and CD34+ expansion in vitro.

Throughout the course of the expansion, differences in cell count and the formation of colonies between conditions were visually observed (Figures 2A and 2B). Just by visual observation, no differences in cell phenotype were observed by direct visual inspection.

Therefore, the cells were quantified with FACS analysis. The cells were characterized by Lin- CD34+CD38CD45RA-CD90+CD49F+ and Lin-CD34+ (Figures 3A-3D). For the single cell HSC expansion, the expansion media used herein yielded the highest HSC count with a maximum of 13,343 (mean: 3,985 + 4,516 cells, Figure 3C). This condition was statistically significantly higher than all other conditions except when UM171 was replaced by SRI in the expansion media (p = 0.2204, Figure 3C). Statistical analysis for percentage of live cells that were HSCs were similar to results found for the HSC count, with an average percentage of 16.8 + 17.1% (Figure 3C). For the 5 and 25 cell expansion, the expansion media used herein yielded the highest HSC count and percentage, and differed statistically significantly from all other conditions (Figure 3C). The significant variation observed in the data from the single cell expansion could be explained by the fact that each single cell is developing in a different environment subjected to paracrine signaling.

In the expansion of CD34+ cells, more variability than in HSCs were observed, possibly due to the fact that CD34+ cells are a much more heterogeneous population because they include HSCs and all of their different progenitors (Figure 3D). There were no statistically significant differences in CD34+ count or percentage in the single and 5 cell expansions.

However, replacing the UM171 with SRI yielded the highest cell count and percentage in the 25 cell expansion at 101,063 + 4,154 cells and 47.6 + 6.1% (Figure 3D). Overall, the results indicated that the expansion media used herein yielded the most robust expansion and was most successfully able to maintain the initial cell profile of the HSCs throughout the course of the expansion, yielded the greatest number of cells and highest degree of homogeneity of starting cells at the end of the expansion protocol,

Differentiation of expanded cells

In order to determine the differentiation ability of expanded cells, cells were subjected to differentiation protocol after expansion. Within the monocyte differentiation phase (day 14 - 28), cells began adhering to the plate surface (Figure 4A).

In FACS analysis, it was found that for differentiating cells, the expression of CD34 was decreasing continuously overall (Figure 6A). In the differentiating cells, a very abrupt decrease was observed in CD34 expression in the sHSCs from day 14 to day 21, which is when the media was changed expansion media to MonoMedia (or first differentiation media) (Figure 6A). While the sCD34 cell's expression of this marker decreased more gradually. It is worth noting that the variability in sCD34 cells were much larger than the sHSCs, again, possibly due to the fact that they are a much more heterogeneous population. However, in the control cells which were maintained in the expansion media, the decrease in expression of CD34 for both sCD34 and sHSCs was much more gradual (Figure 6A). In spite of maintaining the cells in expansion media, we suspect there is some exhaustion in the sternness of the cells. This exhaustion seems to appear first in the sCD34 cells, once again due to the fact that they are a much more heterogeneous population containing many progenitors in addition to stem cells.

CD14CD16+ is a phenotypic characterization for monocytes, and a stark difference was observed in its expression between control cells maintained in expansion media and differentiating cells (Figure 6A). In the differentiating cells, about a 20% expression of CD14CD16+ was observed at day 21 and this value continuously increases to about a 50% expression at day 42 (Figure 6A). This indicates that the differentiating cells are adopting monocyte like characteristics. However, in the control cells maintained in the expansion media, there is essentially 0% expression of CD14CD16+ throughout the entirety of the protocol (Figure 6A).

CD1 lb and CD33 are both markers of microglia, and CD33 is relevant to many nervous system disorders (Muffat et al., 2016). In the differentiating cells, expression of CD1 lb was already relatively high at about 25% on day 21 for both sHSCs and sCD34 cells (Figure 6A). Expression of CD1 lb continued to increase and reaches about a maximum of 75% at day 35 (Figure 6A). The CD33 expression in the differentiating cells increased abruptly between day 21 and day 28, and remained relatively constant at nearly 100% expression for the rest of the protocol for both sHSCs and sCD34 cells (Figure 6A). In the control (expansion media) however, the expression of CDl lb on day 21 was essentially 0% and the expression does not seem to increase much until day 35 where they collectively reach around 30% expression (Figure 6A). However, the expression of CD33 in the control cells was very similar to the expression pattern of the differentiating cells (Figure 6A). Overall, the increase in both of these markers in the control cells may be due to spontaneous differentiation and/or exhaustion of sternness, due to these markers being expressed by a broad range of cell types in the blood.

A phenotypic characterization that is used to identify microglia is the ratio of expression between CCR2 and CX3CR1 (Ohgidani et al., 2014). It has been shown that there is a distinct difference in expression of these two markers between monocytes and resident microglia - monocytes show CCR2^high and CX3CRl^low while resident microglia show CCR2^low and CX3CRl^hlgh. Because of this, the ratio between CX3CR1/CCR2 was used as an indicator of microglia induction. It was observed that the ratio of these markers increased through the course of the microglia differentiation phase (Fig 5B). On day 21, the ratio of CX3CR1/CCR2 was 0.2 + 0.1 and by day 42, the ratio increased to 1.0 + 0.6 (Figure 6B).

Throughout this protocol, it was observed that the sHSCs appeared to be more branched and had a more microglia- like morphology compared to the sCD34 cells which had a greater number of rounded cells and fewer branched cells compared to the sHSCs (Figures 3A- 3D).

Phenotypic and functional characterization of microglia-like cells

Immunocytochemistry

IBA1 is a protein that is specifically expressed by macrophages and microglia. In order to immunophenotypically characterize the microglia-like cells, the differentiated sHSCs and sCD34 cells were stained using an antibody for IBA1. It was observed that the control cells that were kept in expansion media had very insignificant staining for IBA1 whereas the differentiated cells had high expression of IB A 1 (Figure 7A).

Phagocytosis Assay Because phagocytosis is one of the central functions of microglia, the phagocytic ability of our microglia-like cells were verified. Phagocytosis was tested by first treating the cells for 24 hours with fluorescent latex beads and then observed the internalization of these beads via microscope. It was found that there was significant internalization of the fluorescent beads by the cells in culture (Figure 7B). Afterwards, these cells were fixed and used for immunocytochemistry which showed co-localization of beads with IBA1 staining (Figure 7C).

Cytokine Array

In order to distinguish between the different cytokine profiles that are observed in activated and non-activated microglia, whether the supernatant of the microglia-like cells contain a typical level of cytokines and chemokines in these states was tested. The microglia- like cells were stimulated with IFN-gamma and LPS in order to induce an activated state. As expected, an increase in the level of IFN-gamma between the stimulated and non-stimulated control cells was observed (Figures 8A-8B). We also observed a significant increase in CCL2 (2.2x), MIP-la/b 2.7x), CXCL10 (5.8x), IL-6 (3.3x), Serpin El (1.5x) and TNF-a (1.6x) and a decrease in CXCLQ (0.8x), GM-CSF (0.9x), IL-8 (0.7x) and MIF (0.6x) (Figure 8B). In particular, CXCL10, CCL2, IL-6 and MIP- la/b had 2-6 times higher expression in the stimulated cells (Figure 8B).

Direct differentiation of CD34+ cell population

Since it was previously determined that expanded sHSCs and sCD34 cells

differentiated successfully, it was to see if unexpanded cells could also be differentiated because in some instances, expansion may not be necessary. The results from FACS analysis show a very similar trend as the data from differentiation following expansion. A depleted expression of CD34 was observed over time, and an overall increase in CD33, CD1 lb and CD14CD16+ (Figure 9A). The results of the phagocytosis assay indicated that the

differentiated cells successfully phagocytose and internalize the fluorescent latex beads (Figure 9B). The induced microglia-like cells were observed to stain positively for IBA1 which was consistent with our former results (Figure 9B). The cytokine array was also repeated. Increases between the untreated control cells and IFN-gamma and LPS stimulated cells were observed and were relatively consistent with that of the results from the differentiation following expansion. As expected, an increase in the level of IFN-gamma was observed again between the stimulated and non-stimulated control. A significant increase in CCLl (5.2x), CCL2 (1.6x), MIP-la/b (4.0x), CCL5 (7.4x) CXCL1 (1.2x), CXCL10 (20.9x), GM-CSF (l.lx), IFN-gamma (11.4x), IL1-RA (1.8x), IL-6 (12.4x), IL-8 (l.lx), MIF (0.7x), Serpin El (3.1x) and TNF-a (9.1x) and no decreases in expression in any of the cytokines (Figures 9D and 9E) were observed. However, the overall trend in the change of cytokines expression was similar to the microglia-like cells derived from differentiation preceded by expansion. In particular, we observed a high increase in the expression of IFN-gamma, CXCL10 and IL-6.

Re-plating

In order to determine the conditions that yielded the best re-plating capabilities of the microglia-like cells, re -plating in wells of a 48 flat bottom plate was tested. The cells were re- plated in untreated wells, wells treated with 0.1% gelatin, and wells treated with fibronectin. Through observation via microscope, it was determined that 0.1% gelatin coated wells allowed for the best re-plating abilities, while maintaining the initial cell morphology.

Discussion

Through the experimentation, a robust protocol for expansion of HSCs and CD34+ cells, as well as a robust protocol to differentiate HSCs and CD34+ into microglia-like cells in vitro, was developed. It was demonstrated that incubating HSCs and CD34+ with expansion media yields successful clonal expansion while effectively maintaining the initial cell profile. By treating CD34+ cells and HSCs with IMDM supplemented with M-CSF, hSCF, Flt-3, and IL-3, differentiation towards monocytes was successfully induced. Following the monocyte differentiation, differentiation into microglia-like cells was successfully induced by treating the cells with RPMI supplemented with GM-CSF and IL-34.

HSCs are a notoriously rare population of multipotent stem cells that give rise to all the blood cells in the body. And due to the small number of HSCs and the difficulty of expanding them ex vivo, their potential for their widespread use in human transplantation has been stifled. Thus, developing a protocol that is able to expand them while maintaining their sternness would be of great value. One compound that has been found to be relatively successful in maintaining the expansion of HSCs while maintaining their cell profile ex vivo is a small molecule called UM171 (Fares et al., 2014). Another compound that has been identified is called StemRegenin or SRI, which also promotes ex vivo expansion and maintenance of HSCs (Boitano et al., 2010). However, neither of these compounds were ideal in their ability to maintain and expand HSCs so a more effective and robust media was developed. The media developed herein, the expansion media, was found to be more effective in maintaining the cell profile of HSCs throughout the course of expansion, which could further the accessibility of HSC transplantation for various therapies.

Though a few protocols for differentiating iPS cells and monocytes into microglia-like cells exist, there are none yet established to effectively derive microglia-like cells from HSCs. This would be a valuable task to achieve because of the potential of HSC transplantation to become a therapy for various neurological diseases. In 2016, an effective protocol for differentiating microglia-like cells from human iPS cells was published (Muffat et al., 2016). Through culturing iPS cells in their unique media, Muffat et al., (2016) were able to induce differentiation of microglia-like cells with appropriate morphology, cytokine profiles and immunophenotypic characteristics. This differentiation protocol takes over 2 months at 74 days, which is very laborious. In 2014, Ohgidani et al., developed a protocol for the direction differentiation of human monocytes into microglia-like cells by treating human monocytes with a cocktail of IMDM supplemented with GM-CSF and IL-34. This protocol developed by Ohgidani et al., (2014) however, requires an extremely large number of cells at a density of 4 x 10⁵ monocytes and is thus a difficult protocol to use in practice and to establish scalability. The protocol described herein successfully takes a small number of HSCs, expands them and induces microglia-like cells. This protocol improves on the efficiency of the Muffat et al., (2016) by decreasing the protocol down to 42 days. Further, in this protocol differentiation was accomplished while using much fewer cells than in the protocol of Ohgidani et al., (2014). A robust protocol for the differentiation of HSCs and CD34+ cells into microglia-like cells, creates a scalable platform that mimics in vivo processes. This can be used not only to understand these processes, but also as a platform to model diseases and screen small molecules that could potentially act as drugs for microglia-related diseases.

Throughout the course of the differentiation protocol, the appearance of microglia-like morphology was observed. However, at the end of the protocol, it was crucial for to determine if the functionality of the differentiated cells was comparable to that of microglia. To accomplish this, phagocytosis and cytokine assays, which were performed by Muffat et al., (2016) and Ohgidani et al., (2014) as well, were performed. In the phagocytosis assay, it was determined that the microglia-like cells successfully phagocytosed the fluorescent beads which were visualized using fluorescent microscopy. This was an important functionality to test because one of the central roles of microglia in the CNS is to phagocytose foreign materials and damaged neurons. The cytokine assay described above proved to be very informative about the properties of the microglia-like cells. A marked increase in CCL2, MIP-la/b, CXCL10, IL-6, TNF-a, and Serpin El and a decrease in CXCLQ, GM-CSF, IL-8, and MIF was observed. In particular, CXCLIO, CCL2, IL-6, and MIP-la/b had 2-6 times higher expression in the stimulated cells. A study published by Clarner et al., (2015) showed that CXCLIO is heavily involved in the initiation of microglial activation in the cuprizone model, which is a mouse model for multiple sclerosis. In another study published by Lannes et al., (2017), where researchers tested the effects of Japanese encephalitis virus (JEV) on primary human microglial cultures due to severe JEV infections causing serious inflammation in the CNS in patients. They found that when treated with inactivated JEV, the primary microglial cultures adopted a pro-inflammatory state where they observed an increase in levels of CXCLIO and CCL9 among other cytokines. There is a large portion of the HIV positive population that is affected by HIV-associated neurocognitive disorders, or HAND (Faissner et al., 2014). This is induced by the infiltration of infected monocytes into the CNS, where they activate microglia, resulting in neurodegeneration. Thus, Faissner published a study which determined that enhanced secretion of CXCLIO, and CCL2 in addition to other cytokines were indicative of microglial activation induced by HIV-transduced monocytoid cells. They also found that the cerebrospinal fluid (CSF) of HIV positive patients had increased expression of CXCLIO, and IL-6 which were correlated with neurodegeneration. Since neurodegeneration and dementia often are more prevalent in older age groups, Ye et al., (1999) studied microglial cytokine profiles in aged mice and found that IL-6 expression was increased in the microglia of older mice. Additionally, in a study done by McManus et al., (1998) it was found that stimulating human fetal microglial cultures with inflammatory stimuli IFN-gamma or LPS caused an increase in the level of MIP-la and MlP-lb. These data together suggest that CXCLIO, CCL2, IL-6, and MIP-la are involved in microglia related neuroinflammation and neurotoxicity in a variety of neurological disorders.

The cytokine array that was performed proved to be very informative about the properties of our microglia-like cells. A marked increase in CXCLIO, CCL2, IL-6 and MIP- la/b upon stimulation with IFN-gamma and LPS was observed. A study published by Clarner et al., (2015) showed that CXCLIO is heavily involved in the initiation of microglial activation in the cuprizone model, which is a mouse model for multiple sclerosis. In another study published by Lannes et al., (2017), where researchers tested the effects of Japanese encephalitis virus (JEV) on primary human microglial cultures due to severe JEV infections causing serious inflammation in the CNS in patients. It was found that when treated with inactivated JEV, the primary microglial cultures adopted a pro-inflammatory state where they observed an increase in levels of CXCLIO and CCL9 among other cytokines. There is a large portion of the HIV positive population that is affected by HIV-associated neurocognitive disorders, or HAND (Faissner et al., 2014). This is induced by the infiltration of infected monocytes into the CNS, where they pathologically activate microglia, resulting in neurodegeneration. Thus, Faissner et al., (2014) published a study which determined that enhanced secretion of CXCLIO, and CCL2 in addition to other cytokines were indicative of microglial activation induced by HIV- transduced cells. It was also found that the cerebrospinal fluid (CSF) of HIV positive patients had increased expression of CXCLIO, and IL-6 which were correlated with neurodegeneration. Since neurodegeneration and dementia often are more prevalent in older age groups, Ye et al., (1999) studied microglial cytokine profiles in aged mice and found that IL-6 expression was increased in the microglia of older mice. In a study done by McManus et al., (1998) it was found that stimulating human fetal microglial cultures with inflammatory stimuli IFN-gamma or LPS caused an increase in the level of MIP-la and MlP-lb. Additionally, increases in the cytokines TNF-a and Serpin El were observed. TNF-a is a cytokine commonly known to be involved in neurotoxicity and neuroinflammation. While this cytokine performs many homeostatic roles, it is also involved in the pathologies of many neurological diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, traumatic brain injury and ischemia (Olmos and Llado, 2014). Though the role of serpin El in the CNS was not clear, Jeon et al., (2012) discovered that it's secretion was increased in murine microglia and astrocyte cultures when they were stimulated with LPS and IFN-gamma. The results indicate that serpin El may be involved in microglial migration and phagocytosis, and may play a role in disease states (Jeon et al., 2012). These data together suggest that the cytokines which we observed increased expression of in our IFN-gamma and LPS stimulated cells including CXCLIO, CCL2, IL-6 and MIP-la, are involved in microglia related

neuroinflammation and neurotoxicity in a variety of neurological disorders.

The literature and the cytokine assay described herein indicate that there are a variety of different neurological disorders that attribute their deleterious effects to microglia. Thus, it is important to have a robust platform on which large masses of different drugs and compounds can be tested in order to ultimately develop a therapy for these microglia related neurological disorders. Through the robust differentiation protocol outlined herein that only requires an initial very few HSCs or CD34+ cells, this much necessary platform could be developed. In terms of practicality, this protocol is ideal for such uses as it requires very few cells, but is able to produce a large number of functional micro glia-like cells.

Another benefit to inducing differentiation of HSCs into microglia-like cells is that it mimics the in vivo process that occurs following bone marrow transplantation (Priller et al., 2001). Currently, HSC or bone marrow transplantation is the only effective cellular transplantation that is commonly used in medical practices. It has been found that following bone marrow transplantation, HSCs and their progenitors get recruited to the brain and the bone marrow derived microglia carry out standard microglial functions within the CNS. By creating a robust protocol for the differentiation of HSCs and CD34 cells into microglia-like cells, we are creating a platform that mimics an in vivo processes on which various

experiments can be performed.

Another application for this differentiation protocol could be utilizing HSC

transplantation as a possible therapy for neurological disorders. Priller et al., (2001) conducted a study investigating the delivery of gene-modified HSCs into the CNS and discovered microglial engraftment in the CNS. They found that in mouse lesion models of stroke, cholinergic fiber degeneration and motor neuron injury, genetically modified green fluorescent protein (GFP) HSCs migrated to the site of CNS injury, and differentiate into microglia (Priller et al., 2001). Since a large challenge in treating neurological disorders is the presence of the blood brain barrier, HSCs may be promising gene modification delivery system into the CNS in the future. In order to further develop and grow this possible therapeutic method, it will be important to have an ex vivo model of this process of HSC to microglia differentiation and the protocol outlined herein could be instrumental in this field of research.

To this end, it is important to continue performing functional, phenotypic and expression assays on these differentiated cells and compare them to resident microglia. It would also be beneficial to investigate how these induced microglia-like cells interact with neuronal cells extracted from a human brain. Overall, the methods of generating microglia-like cells described herein are promising and have great potential to contribute to the advancement of studying microglia's function and its involvement in many neurological disorders. With the growth of knowledge that our protocol could contribute to, new therapies and interventions for these disorders may be discovered.

REFERENCES

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Djukic M, Mildner A, Schmidt H, Czesnik D, Bru^'ck E, Priller J, Nau R, Prinz M Circulating monocytes engraft in the brain, differentiate into microglia and contribute to the pathology following meningitis in mice. Brain 129:2394-2403 (2006).

Dolmetsch R, Geschwind DH The human brain in a dish: The promise of iPSCderived neurons. Cell 145: 831-834 (2013).

Fares I, Chagraoui J, Gareau Y, Gingras S, Ruel R, Mayotte N, Csaszar E, Knapp DJ, Miller P, Ngom M, Imren S, Roy DC, Watts KL,Kiem HP, Herrington R, Iscove NN, Humphries RK, Eaves CJ, Cohen S, Marinier A, Zandstra PW, Sauvageau G. Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self- renewal. Science, 345: 1509-1512 (2014).

Faissner S I, Ambrosius B l, Schanzmann Kl, Grewe B2, Potthoff A3, Miinch J4, Sure U5, Gramberg T6, Wittmann S6, Brockmeyer N3, Uberla K2, Gold Rl, Grunwald T7, Chan A8. Cytoplasmic HIV-RNA in monocytes determines microglial activation and neuronal cell death in HIV- associated neurodegeneration. Exp Neurol., 261:685-697 (2014).

Jeon H, Kim J, Kim J, Lee W, Lee M, Suk K Plasminogen activator inhibitor typel regulates microglial motility and phagocytic activity. Journal of Neuroinflammation 9: 149 (2012).

Lannes N, Neuhaus V, Scolari B, Kharoubi-Hess S, Walch M, Summerfield A, Filgueira L. Interactions of human microglia cells with Japanese encephalitis virus. Virol J., 14: doi: 10.1186/sl2985-016-0675-3 (2017).

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Example 2. Using The Induced Microglia-like Cells as A Platform for Drug Screening

Induced microglia-like cells were treated with JAK2 inhibitor TG101209, concomitant to treatment with LPS and IFN-γ for 24 hours (Figure 10A). JAK2 inhibition has been described to attenuate inflammatory markers in microglia of Alzheimer's disease animal models (e.g., in Jones et al., Volume 36, Issue 10, Pages 2716-2724, Neurobiology of Aging, 2015, incorporated herein by reference). Corroboratively, application of TG101209 on LPS and IFN-y treated microglia-like cells prevented an increase in the TNF-a concentration in the cells' supernatant (Figure 10B). Moreover, analysis of treated cells following fixation and staining with IBA1 antibody showed detectable differences in cellular area (Figure IOC), aspect ratio (Figure 10D), and mean fluorescence intensity (MFI, Figure 10E). This indicates potential for adapting this protocol for utilization as an alternative method of screening new therapeutic targets using patient-derived microglia-like cells.

All publications, patents, patent applications, publication, and database entries (e.g., sequence database entries) mentioned herein, e.g., in the Background, Summary, Detailed Description, Examples, and/or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

Articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes "or" between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

It is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term

"comprising" is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

Where websites are provided, URL addresses are provided as non-browser-executable codes, with periods of the respective web address in parentheses. The actual web addresses do not contain the parentheses.

In addition, it is to be understood that any particular embodiment of the present disclosure may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the disclosure, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

Claims

CLAIMS What is claimed is:

1. A method of generating a microglia-like cell, the method comprising:

(i) differentiating a cell that is an induced pluripotent cell (iPS), a hematopoietic stem cell (HSC), or a CD34+ cell into a monocyte; and

(ii) differentiating the monocyte obtained in (i) into a microglia-like cell.

2. The method of claim 1, wherein the differentiating in (i) comprises contacting the cell with an effective amount of macrophage colony- stimulating factor (M-CSF), stem cell factor (hSCF), receptor-type tyro sine-protein kinase FLT3 (Flt-3), interleukin-3 (hIL-3), or combinations thereof.

3. The method of claim 1 or claim 2, wherein the differentiating in (ii) comprises contacting the monocyte with an effective amount of granulocyte macrophage colony- stimulating factor (GM-CSF) and interleukin-34 (IL-34).

4. The method of any one of claims 1-3, further comprising expanding the cell prior to the differentiating of (i) to produce a population of the cell.

5. The method of claim 4, wherein the expanding comprises contacting the cell with an effective amount of SCF, thrombopoietin (TPO), IL3, Flt3L, A83-01, Pomalidomide, and UM171.

6. The method of any one of claims 1-5, wherein the cell is an iPS.

7. The method of any one of claims 1-5, wherein the cell is a HSC.

8. The method of claim 7, wherein the HSC has a surface expression profile of lineage - CD235a-CD34+CD38-CD45RA-CD90+.

9. The method of any one of claims 1-5, wherein the cell is a CD34+ cell.

10. The method of claim 9, wherein the CD34+ cell is generated from an iPS.

11. The method of claim 9 or claim 10, wherein the CD34+ cell has a surface expression profile of CD34+CD45+.

12. The method of any one of claims 1-11, wherein the monocyte expresses any one of CD14, CD16, CDl lb, and/or CD33.

13. The method of any one of claims 1-12, wherein the microglia-like cell expresses IBAl.

14. The method of any one of claims 1-13, wherein the microglia-like cell has an increased CX3CR1/CCR2 expression ratio compared to the monocyte.

15. The method of claim 14, wherein the CX3CR1/CCR2 expression ratio is increased by at least 50% compared to the CX3CR1/CCR2 expression ratio of monocytes.

16. The method of claim any one of claims 4-15, wherein the population of the cell is used to generate microglia-like cells.

17. The method of claim 16, wherein at least 30% of the population of the cell are differentiated into monocytes after the differentiating in (i).

18. The method of any one of claims 1-17, wherein the differentiating in (i) is carried out for no more than 14 days.

19. The method of any one of claims 1-18, wherein the differentiating in (ii) is carried out for no more than 14 days.

20. The method of any one of claims 4-19, wherein the expanding is carried out for no more than 14 days.

21. A method of generating a microglia-like cell, the method comprising:

(i) expanding a cell that is an induced human pluripotent cell (iPS), a hematopoietic stem cell (HSC), or a CD34+ cell to generate a population of the cell; (ii) differentiating the population of the cell obtained in (i) into monocytes; and

(iii) differentiating the monocytes obtained in (ii) into microglia-like cells.

22. The method of any one of claims 1-21, wherein the method is carried out in vitro.

23. The method of any one of claims 1-21, wherein the method is carried out ex vivo.

24. The method of any one of claims 1-23, wherein the cell is a mammalian cell.

25. The method of claim 24, wherein the cell is a human cell.

26. A microglia-like cell generated by the method of any one of claims 1-25.

27. The microglia-like cell of claim 26, wherein the microglia-like cell secretes a cytokine upon stimulation.

28. The microglia-like cell of claim 27, wherein the cytokine is selected from the group consisting of: CCL2, MIP-la/b, CXCLIO, IL-6, TNF-a, Serpin El, and combinations thereof.

29. The microglia-like cell of any one of claims 26-28, wherein the microglia-like cell is phagocytic.

30. The microglia-like cell of any one of claims 26-29, wherein the microglia-like cell expresses IBA1 on its surface.

31. The microglia-like cell of any of claims 26-30, wherein the microglia-like cell has comparable biological activity as a natural microglia.