Rapid and deterministic generation of microglia from human pluripotent stem cells

Abstract

The present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeting the insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor locus; and b) targeting the insertion of the coding sequence of the transcription factor PU.1 (SEQ ID NO:1) into a second genomic safe harbor locus, wherein the gene is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; expressing PU.1 (SEQ ID NO:2); and culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that mimics signaling during at least one stage of microglial embryonic development or adult microglial proliferation, differentiation, or polarization. In addition, the present invention relates to the microglia obtained by the method of the present invention and various uses thereof.

Description

Technical Field

[0001] The present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: targeting the insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor locus; and targeting the insertion of a coding sequence of the transcription factor PU.1 into a second genomic safe harbor locus, wherein the gene is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; expressing PU.1; and culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that recapitulates the signaling during at least one stage of embryonic development of microglia or adult microglial proliferation, differentiation or polarization. Furthermore, the present invention relates to microglia obtained by the method of the present invention and their various uses. Background Art

[0002] Microglia are resident immune cells in the central nervous system (CNS) [Schafer et al., 2015]. They originate from early yolk sac macrophages that arise during the first wave of primitive hematopoiesis in early embryonic development. Once the circulatory system is established, primitive yolk sac macrophages spread through the bloodstream to populate the developing CNS. In contrast to tissue-resident macrophages in other organs, microglia are not replaced by fetal monocytes at later stages of embryonic development [McGrath et al., 303; Ginhoux et al., 2010; Gomez Perdiguero et al., 2015]. After the microglial population is established during early embryonic development, microglia self-maintain throughout life by local proliferation rather than being replaced by bone marrow-derived cells [Réu et al., 2017]. Microglia are evenly distributed throughout the brain and spinal cord and play a key role in the development, maintenance, plasticity and defense of the CNS [Schafer et al., 2015]. In a healthy CNS, "resting" homeostatic microglia are highly branched cells with small cell bodies and fine cell processes. These microglial processes are motile and continuously sample their environment to scan for signals of internal or external danger, such as invading pathogens or signals locally produced by damaged or dying cells. Detection of these signals leads to microglial activation, which includes profound changes in microglial morphology, gene expression and function. After activation, microglia retract their processes and revert to an amoeboid appearance. They actively migrate towards CNS lesions following chemotactic gradients and secrete inflammatory cytokines.

[0003] Communicate with neurons, other glial cells, and peripheral immune system cells through a large number of cell surface receptors, including neurotransmitter and cytokine receptors [Kettenmann et al., 2011]. Given their multifunctionality and their unique position as representatives of the immune system in the healthy CNS, it is not surprising that microglia are involved in the onset and progression of many neurological diseases [Ranshoff et al., 2016].

[0004] Recently, single-cell transcriptomic analysis of microglia in mouse models of Alzheimer's disease (AD) and other neurodegenerative diseases, including aging, amyotrophic lateral sclerosis, or tauopathy-related frontotemporal lobar degeneration (FTLD-tau), has revealed a pro-inflammatory transcriptomic signature in a small subset of microglia, which is termed the microglial neurodegenerative phenotype (MGnD) [Krasemann et al., 2017] or disease-associated microglia (DAM) [Keren-Saul et al., 2017]. The conversion of microglia from a homeostatic to a disease-associated phenotype is thought to be a response to altered brain homeostasis in neurodegeneration and depends on a unique temporally and spatially controlled transcriptional program [Krasemann et al., 2017; Keren-Shaul et al., 2017; Butovsky et al., 1998]. In most cases, it remains unclear whether these cells have protective or disease-inducing / proliferative functions. Studies of human microglia in vitro and in vivo, in health and disease, will facilitate the identification of factors associated with their beneficial and harmful functions, as well as the development of strategies to restore the homeostatic microglial signature or induce the DAM microglial signature. This could allow us to target microglia for the treatment of neurodegenerative diseases.

[0005] The isolation or in vitro derivation of many human cell types remains challenging and inefficient. In particular, cells of the human CNS, including microglia, are particularly difficult to obtain. In the past, inefficient isolation from neurosurgical samples or postmortem brain tissue represented the only access. Human pluripotent stem cells (hPSCs) represent an unlimited and renewable source from which, in theory, all cell types of the human organism can be generated [Thomson et al., 1998]. The groundbreaking discovery that human skin fibroblasts can be readily converted into human induced pluripotent stem cells (hiPSCs) that display the same properties as embryonic stem cells allows the generation of autologous and customized cell types for applications in regenerative medicine. For several key applications, including disease modeling, drug discovery, and cell transplantation, the large-scale manufacture of mature human cell types from hPSCs is required. Recently, the first hPSC differentiation protocol for the generation of microglia was published. This protocol was based on initially formed embryoid bodies (EBs) cultured for several months in the same “glial differentiation medium, the component concentrations of which were adjusted to match those of human cerebrospinal fluid”, supplemented with interleukin (IL)-34 and colony-stimulating factor 1 (CSF-1) [Muffat et al., 2016]. This pioneering literature provided a detailed medium composition for the final maturation and maintenance of human microglia. However, the long duration of this protocol, the uncertain initial differentiation steps (i.e., the EB-based intermediate steps that hardly follow embryonic principles), and the need for several mechanical operation steps for cell purification may impede the widespread application of this protocol. Subsequently, several other groups verified the generation of microglia-like cells from hPSCs by similar but different classical differentiation methods [Abud et al., 2017; Takata et al., 2017; Haenseler et al., 2017; Pandya et al., 2017; Douvaras et al., 2017]. However, the in vitro derivation of specific human cell types, including microglia, in the quantities and purities required for downstream applications remains challenging, and alternative methods are currently being sought [Cohen et al., 2011]. Compared with traditional differentiation, a recent production strategy is direct cell reprogramming [Ladewig et al., 2013]. This strategy refers to the direct conversion of any cell type (usually skin fibroblasts) into another cell type without going through a pluripotent intermediate. Although providing a rapid route to produce cells from readily available cell types, the yields and purities of the required cell populations are still low and insufficient [Zhang et al., 2013]. Recently, a third approach, termed “forward programming”, has been proposed for manufacturing mature human cell types at unprecedented speeds and efficiencies [Zhang et al., 2013].

[0006] As a method for directly converting pluripotent stem cells (including hPSCs) into mature cell types, forward programming has been considered a powerful strategy for deriving human cells. It involves the forced expression of key lineage transcription factors (or non-coding RNAs, including lncRNAs and microRNAs) in order to convert stem cells into specific mature cell types. Currently available forward programming protocols are mainly based on lentiviral transduction of cells, which results in diverse expression or complete silencing of randomly inserted induction cassettes. This leads to the need for additional purification steps to isolate the subpopulation expressing the desired transcription factor. Thus, there is clearly a need for further improvement of these methods.

[0007] Any improvement to the method must ensure that stable transcription of genetic material such as transgenes contained within the induction cassette is resistant to silencing and other negative integration site-related effects. Silencing can be caused by a variety of epigenetic mechanisms including DNA methylation or histone modification. Using existing art methods based on lentiviral transduction, the cells obtained are a heterogeneous population in which the transgene is expressed fully, partially, or silenced. Clearly, this is undesirable for many applications. Viral vectors have a tendency to integrate their genetic material into transcriptionally active regions of the genome, thereby increasing the likelihood of oncogenic events due to insertional mutagenesis. For many applications, it is necessary to control the transcription of the inserted genetic material in the cell so that the induction cassette can be switched on as needed and transcribed at a specific level (including high levels). This cannot be achieved if the insertion of the induction cassette into the genome is random.

[0008] This problem has been addressed in some publications, namely that microglia are involved in some severe diseases and are so entangled with brain tissue that their isolation from live tissue remains elusive. To overcome this problem, human stem cells are used to generate microglia or microglia-like cells by, for example, defined culture conditions [Muffat et al., 2016] or co-culture with stem cell-derived neurons [Haenseler et al., 2017; Takata et al., 2017]. These methods rely only on exposure to growth factors and cytokines to differentiate stem cells into microglia.

[0009] In addition, the need for such special cell types is huge because they play important roles in almost all central nervous system diseases, including neurodegenerative diseases, neuroinflammatory or autoimmune diseases, autoantibody-mediated encephalitis or infectious diseases, neurovascular diseases, stroke, traumatic brain injury, and cancer. However, the exact mechanisms of their roles in different diseases are still unclear. Consistent with the prior art, stem cell-derived microglia do reproduce the original patient disease phenotypes [Muffat et al., 2016; Abud et al., 2017; Takata et al., 2017]. Based on this, the huge scientific gap in the involvement of microglia in certain diseases can be overcome by generating microglia from stem cells. However, the traditional protocols for differentiating stem cells are very time-consuming and the results are not convincing.

[0010] Therefore, the inventors of the present invention have developed a rapid method for generating microglia from stem cells by using an induction cassette stably introduced into the stem cell genome while being able to control the transcription of the induction cassette and thereby control the inserted transcription factors. The potential of these transcription factors as reprogramming factors for microglia generation was previously unknown and represents the unique knowledge of the inventors. This enables them to generate a pure population of microglia expressing all surface markers and RNAs observed in the native microglia population. In addition, the method can be used to differentiate microglia from human iPS cells of patients with neurodegenerative diseases, thereby enabling the analysis of cell populations that would otherwise remain completely inert to medical examination. Therefore, there is a strong need to manufacture mature human microglia from easily accessible sources. Therefore, the technical problem faced by the present application is to meet these needs. By providing the embodiments reflected in the claims, described in the specification, and shown in the examples and drawings given below, the above technical problems are solved. Summary of the Invention

[0011] The inventors of the present invention have developed a method for generating microglia from stem cells.

[0012] The present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeting the insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor site; and b) targeting the insertion of the coding sequence of the transcription factor PU.1 (SEQ ID NO:1) into a second genomic safe harbor site, wherein the gene is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; expressing PU.1 (SEQ ID NO:2); and c) culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that reproduces the signaling during at least one stage of embryonic development or adult microglia proliferation, differentiation, or polarization of microglia.

[0013] In one embodiment of the method of the present invention, the at least one growth factor or small molecule is selected from activin A (SEQ ID NO:7), BMP4 (SEQ ID NO:8), FGF (SEQ ID NO:9), VEGF-A (SEQ ID NO:10), LY294002, CHIR99021, SCF (SEQ ID NO:11), IL-3 (SEQ ID NO:12), IL-6 (SEQ ID NO:13), CSF1 (SEQ ID NO:14), IL-34 (SEQ ID NO:15), CSF2 (SEQ ID NO:16), CD200 (SEQ ID NO:17), CX3CL1 (SEQ ID NO:18), TGFβ1 (SEQ ID NO:19), and IDE1.

[0014] In another embodiment of the method of the present invention, the at least one growth factor is CSF1 (SEQ ID NO:14) or IL-34 (SEQ ID NO:15).

[0015] In yet another embodiment of the method of the present invention, the at least one small molecule is CHIR99021, LY294002, or IDE1.

[0016] In still another embodiment of the method of the present invention, the first and second genomic safe harbor sites are different.

[0017] In yet another embodiment of the method of the present invention, the method further comprises the insertion and expression of the coding sequence (SEQ ID NO:3) of the gene of the transcription factor CEBPB.

[0018] In still yet another embodiment of the method of the present invention, the method further comprises the insertion and expression of the coding sequence (SEQ ID NO:4) of the gene of the transcription factor RUNX1.

[0019] In another embodiment of the method of the present invention, the method further comprises the insertion and expression of the coding sequence (SEQ ID NO:5) of the gene of the transcription factor IRF8.

[0020] In yet another embodiment of the method of the present invention, the method further comprises the insertion and expression of the coding sequence (SEQ ID NO:6) of the gene of the transcription factor SALL1.

[0021] In yet another embodiment of the method of the present invention, the transcriptional regulatory protein is a reverse tetracycline trans-activator protein (rtTA) (SEQ ID NO: 20), and its activity is controlled by doxycycline or tetracycline.

[0022] In yet another embodiment of the method of the present invention, the inducible promoter comprises a Tet-responsive element (TRE) (SEQ ID NO: 21).

[0023] In yet another embodiment of the method of the present invention, the first and the second genomic safe harbor loci are selected from the hROSA26 locus (SEQ ID NO: 22), the AAVS1 locus (SEQ ID NO: 23), the CLYBL gene (SEQ ID NO: 24), the CCR5 gene (SEQ ID NO: 25), the HPRT gene (SEQ ID NO: 26), or genes having locus ID 325 on chromosome 8 (SEQ ID NO: 27), locus ID 227 on chromosome 1 (SEQ ID NO: 28), locus ID 229 on chromosome 2 (SEQ ID NO: 29), locus ID 255 on chromosome 5 (SEQ ID NO: 30), locus ID 259 on chromosome 14 (SEQ ID NO: 31), locus ID 263 on chromosome X (SEQ ID NO: 32), locus ID 303 on chromosome 2 (SEQ ID NO: 33), locus ID 231 on chromosome 4 (SEQ ID NO: 34), locus ID 315 on chromosome 5 (SEQ ID NO: 35), locus ID 307 on chromosome 16 (SEQ ID NO: 36), locus ID 285 on chromosome 6 (SEQ ID NO: 37), locus ID 233 on chromosome 6 (SEQ ID NO: 38), locus ID 311 on chromosome 134 (SEQ ID NO: 39), locus ID 301 on chromosome 7 (SEQ ID NO: 40), locus ID 293 on chromosome 8 (SEQ ID NO: 41), locus ID 319 on chromosome 11 (SEQ ID NO: 42), locus ID 329 on chromosome 12 (SEQ ID NO: 43), locus ID 313 on chromosome X (SEQ ID NO: 44).

[0024] In another embodiment of the method of the present invention, the stem cells are pluripotent stem cells, induced pluripotent stem cells (iPSCs), neural progenitor cells, hematopoietic stem cells, or embryonic stem cells (ESCs).

[0025] In yet another embodiment of the method of the present invention, the stem cells are human or murine stem cells.

[0026] The present invention also relates to microglia obtained by any of the methods according to the present invention, preferably, wherein the microglia express at least one microglial surface protein selected from ITGAM (CD11B) (SEQ ID NO: 45), ITGAX (CD11C) (SEQ ID NO: 46), CD14 (SEQ ID NO: 47), CD16 (SEQ ID NO: 48), ENTPD1 (CD39) (SEQ ID NO: 49), PTPRC (CD45) (SEQ ID NO: 50), CD68 (SEQ ID NO: 51), CSF1R (CD115) (SEQ ID NO: 52), CD163 (SEQ ID NO: 53), CX3CR1 (SEQ ID NO: 54), TREM2 (SEQ ID NO: 55), P2RY12 (SEQ ID NO: 56), TM119 (SEQ ID NO: 57) and HLA-DR (SEQ ID NO: 58).

[0027] In a further embodiment of the present invention, the microglia are used for treatment.

[0028] Furthermore, the present invention relates to the use of such microglia according to the present invention for in vitro diagnosis of diseases. Preferably, the diseases are selected from central nervous system diseases, preferably neurodegenerative diseases; more preferably Alzheimer's disease, Parkinson's disease, frontotemporal dementia or amyotrophic lateral sclerosis; neuroinflammatory or autoimmune diseases, preferably multiple sclerosis, autoantibody-mediated encephalitis or infectious diseases, neurovascular diseases; preferably stroke, vasculitis; traumatic brain injury and cancer.

[0029] Furthermore, the present invention relates to the use of such microglia according to the present invention for in vitro culture with brain organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A schematic diagram showing the main pathways for cell manufacturing, which are: reprogramming somatic cells (fibroblasts) into induced pluripotent stem cells (iPSCs) using four defined transcription factors Klf4, Oct4, c-Myc, and Sox2, direct reprogramming of somatic cells into the desired target cell type using defined transcription factors, traditional differentiation methods representing the stepwise conversion from pluripotent stem cells into the desired target cells, and forward programming of hPSCs directly into the target cell type. (Abbreviations: TF = transcription factor, ESC = embryonic stem cell, iPSC = induced pluripotent stem cell (ESC and iPSC are collectively referred to as pluripotent stem cells (PSC)).

[0031] Figure 2 A schematic diagram showing the targeting strategies used in the present invention. The dox-inducible Tet-ON system is targeted to the human ROSA26 locus (CAG-rtTA) and the AAVS1 locus (TRE-EGFP) of hPSCs. (Abbreviations: HAR = homologous arm, Neo = neomycin resistance gene, CAG = constitutive CAG promoter, rtTA = reverse tetracycline-controlled transactivator, Puro = puromycin resistance gene, TRE = inducible Tet-responsive element, EGFP = enhanced green fluorescent protein, SA = splice acceptor, T2A = T2A cleavage site, pA = polyadenylation site).

[0032] Figure 3 A table showing the key transcription factors of the microglial lineage selected as candidate reprogramming factors, the length of their coding sequences, and their sources.

[0033] Figure 4 A schematic diagram showing the donor plasmid, which is generated by molecular cloning and used for genetic modification of ROSA26 GSH or AAVS1 GSH. (Abbreviations: HAR = homologous arm, Neo = neomycin resistance gene, CAG = constitutive CAG promoter, rtTA = reverse tetracycline-controlled transactivator, Puro = puromycin resistance gene, TRE = inducible Tet-responsive element, EGFP = enhanced green fluorescent protein, SA = splice acceptor, T2A = T2A cleavage site, pA = polyadenylation site).

[0034] Figure 5 A schematic diagram showing the microglial forward programming protocol (see Figure 5 A). The time course of cell surface markers expressed on primary macrophages and microglia evaluated by flow cytometry (n = 2 biological replicates) (see Figure 5 B and Figure 5C). Microglia monoculture on day 20: Phase-contrast live images of microglia-like cells and ICC of the microglial characteristic transmembrane protein TMEM119, as dedicated labeled flow antibodies were not available (see Figure 5 D). Microglia / neuron co-culture on day 20: ICC of the intracellular calcium-binding protein IBA1 (also known as AIF1) and the neuronal marker βIII-tubulin (TUBB3) (see Figure 5 E). QPCR (SYBR green) of hiPSCs and microglia in monoculture (day 20). All values were relative to the housekeeping gene GAPDH and normalized to hiPSCs. For the transcripts of SPI1 and CEBPB, two different primer pairs were used (see SEQ ID NOs: 80 - 87; SEQ ID NO: 80: SPI1 total forward primer; SEQ ID NO: 81: SPI1 total reverse primer; SEQ ID NO: 82: SPI1 internal forward primer; SEQ ID NO: 83: SPI1 internal reverse primer; SEQ ID NO: 84: CEBPB total forward primer; SEQ ID NO: 85: CEBPB total reverse primer; SEQ ID NO: 86: CEBPB internal forward primer; SEQ ID NO: 87: CEBPB internal reverse primer) to detect all transcripts (total), or only the transcripts from their respective endogenous loci, but not the AAVS1-targeted transgene (internal). As expected, when transgene expression was turned off (by withdrawing dox on day 10 of the protocol), no differences were detected in the relative expression levels, thus confirming the transgene independence of the cell phenotype (F).

[0035] Figure 6 Immunocytochemistry of the dual-targeted iPS cell line induced with doxycycline for 24 hours is shown. The cells were positive for PU.1 and CEBPB, but negative for OCT4.

[0036] Figure 7 The map of the donor plasmid pUC_AAVS1_p-Resp-(PU.1-CEBPB) (SEQ ID NO: 61) is shown, which is used for genetic modification of the AAVS1 locus and contains the coding sequences of the transcription factors PU.1 and CEBPB.

[0037] Figure 8 The map of the donor plasmid pUC_AAVS1_p-Resp-(PU.1-IRF8) (SEQ ID NO: 62) is shown, which is used for genetic modification of the AAVS1 locus and contains the coding sequences of the transcription factors PU.1 and IRF8.

[0038] Figure 9 The map of the donor plasmid pUC_AAVS1_p-Resp-(PU.1-RUNX1) (SEQ ID NO:63) is shown. This donor plasmid is used for genetic modification of the AAVS1 locus and contains the coding sequences of the transcription factors PU.1 and RUNX1.

[0039] Figure 10 The map of the donor plasmid pUC_AAVS1_p-Resp-(PU.1) (SEQ ID NO:64) is shown. This donor plasmid is used for genetic modification of the AAVS1 locus and contains the coding sequence of the transcription factor PU.1.

[0040] Figure 11 The map of the donor plasmid pUC_AAVS1_p-Resp-(PU.1-SALL1) (SEQ ID NO:65) is shown. This donor plasmid is used for genetic modification of the AAVS1 locus and contains the coding sequences of the transcription factors PU.1 and SALL1.

[0041] Figure 12 The map of the plasmid ROSA-guide A_Cas9n (SEQ ID NO:66) containing the coding sequences of the Cas enzyme and guide RNA A is shown.

[0042] Figure 13 The map of the plasmid ROSA-guide B_Cas9n (SEQ ID NO:67) containing the coding sequences of the Cas enzyme and guide RNA B is shown.

[0043] Figure 14 The map of the donor plasmid pUC_ROSA_n_CAG-rtTA (SEQ ID NO:72) containing the constitutive CAG promoter and rtTA is shown.

[0044] Figure 15 The map of the plasmid pZFN-AAVS1-L_ELD (SEQ ID NO:68) is shown.

[0045] Figure 16 The map of the plasmid pZFN-AAVS1-R_KKR (SEQ ID NO:69) is shown.

[0046] The following abbreviations are used: T2A: T2A peptide (ribosomal skipping signal), puroR: puromycin resistance gene, pA: polyadenylation signal, CAG: constitutive CAG promoter, TRE3GV: Tet-responsive element, HA-R, HA-L: homologous arms (right, left), AmpR: ampicillin resistance gene, ori: replication origin, NeoR: neomycin resistance gene, KanR: kanamycin resistance gene. Detailed implementation mode

[0047] The present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeting the insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor locus; and b) targeting the insertion of the coding sequence of the transcription factor PU.1 (SEQ ID NO: 1) into a second genomic safe harbor locus, wherein the gene is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; expressing PU.1 (SEQ ID NO: 2); and c) culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that recapitulates the signaling during at least one stage of microglial embryonic development or adult microglial proliferation, differentiation, or polarization.

[0048] In one embodiment, the present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeting the insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor locus; and b) targeting the insertion of the coding sequence of the transcription factor PU.1 (SEQ ID NO: 1) into a second genomic safe harbor locus, wherein the gene is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; expressing PU.1 (SEQ ID NO: 2); and c) culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that recapitulates the signaling during at least one stage of microglial embryonic development or adult microglial proliferation, differentiation, or polarization.

[0049] In one embodiment, the present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeting the insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor locus; and b) targeting the insertion of the coding sequence of the transcription factor PU.1 (SEQ ID NO: 1) into a second genomic safe harbor locus, wherein the gene is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; expressing PU.1 (SEQ ID NO: 2); and c) culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that recapitulates the signaling during at least one stage of microglial embryonic development.

[0050] In one embodiment, the present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeting the insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor locus; and b) targeting the insertion of the coding sequence of the transcription factor PU.1 (SEQ ID NO:1) into a second genomic safe harbor locus, wherein the gene is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; expressing PU.1 (SEQ ID NO:2); and c) culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that recapitulates the signaling during at least one stage of microglial embryonic development.

[0051] In one embodiment, the present invention also relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeting the insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor locus; and b) targeting the insertion of the coding sequence of the transcription factor PU.1 (SEQ ID NO:1) into a second genomic safe harbor locus, wherein the gene is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; expressing PU.1 (SEQ ID NO:2); and c) culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that recapitulates the signaling during at least one stage of adult microglial differentiation.

[0052] In a further embodiment, the present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeting the insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor locus; and b) targeting the insertion of the coding sequence of the transcription factor PU.1 (SEQ ID NO:1) into a second genomic safe harbor locus, wherein the gene is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; expressing PU.1 (SEQ ID NO:2); and c) culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that recapitulates the signaling during at least one stage of adult microglial polarization.

[0053] In another embodiment, the present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeting the insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor locus; and b) targeting the insertion of the coding sequence of the transcription factor PU.1 (SEQ ID NO:1) into a second genomic safe harbor locus, wherein the gene is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; expressing PU.1 (SEQ ID NO:2); and c) culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that recapitulates the embryonic development of microglia.

[0054] In a further embodiment, the present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeting the insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor locus; and b) targeting the insertion of the coding sequence of the transcription factor PU.1 (SEQ ID NO:1) into a second genomic safe harbor locus, wherein the gene is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; expressing PU.1 (SEQ ID NO:2); and c) culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that mimics signaling during at least one stage of embryonic development of microglia or adult microglia proliferation, differentiation, or polarization.

[0055] In a further embodiment, the present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeting the insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor locus; and b) targeting the insertion of the coding sequence of the transcription factor PU.1 (SEQ ID NO:1) into a second genomic safe harbor locus, wherein the gene is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; expressing PU.1 (SEQ ID NO:2); and c) culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that mimics signaling during at least one stage of embryonic development of microglia.

[0056] In one embodiment, the invention also relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeting the insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor locus; and b) targeting the insertion of the coding sequence of the transcription factor PU.1 (SEQ ID NO:1) into a second genomic safe harbor locus, wherein the gene is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; expressing PU.1 (SEQ ID NO:2); and c) culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that recapitulates the embryonic development of microglia in vitro.

[0057] As used herein, the term "microglia" refers to a mature cell type that is a distinct cell population of the central nervous system. As defined in comparative anatomy and histology, "microglia are resident tissue cell-type cells and key innate immune effectors of the CNS. They are commonly described as either resting (i.e., ramified) or activated, but these terms do not convey the dynamic remodeling of their fine processes and innate immune surveillance activity. (...) Evidence suggests that early microglia are derived from yolk sac progenitors" (Hagan et al., 2012). This means that microglia are generated early in embryogenesis and remain resident in the brain throughout adulthood.

[0058] As used herein, the term "generation of microglia" means the generation of mature cells (microglia) from stem cells, obtained by any of the methods of the invention described herein.

[0059] As used in the present invention, the term "stem cell" means a cell type that is capable of dividing to produce more cells or capable of developing into cells with a specific purpose. In the present invention, the stem cells used can be pluripotent stem cells. Pluripotent stem cells have the potential to differentiate into almost any cell in the body. There are several sources of pluripotent stem cells. Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early pre-implantation embryo. Induced pluripotent stem cells (iPSCs) are somatic cells that have been genetically reprogrammed into an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defined characteristics of embryonic stem cells. In 2006, it was demonstrated that the introduction of four specific genes encoding transcription factors could convert somatic cells into pluripotent stem cells (Takahashi et al., 2006), but subsequent work has reduced / altered the number of genes required. Certain members of the Oct-3 / 4 and Sox gene families have been identified as potential key transcriptional regulators involved in the induction process. Other genes, including certain members of the Klf family, Myc family, Nanog, and LIN28, can increase the induction efficiency. Examples of genes that can be included in the reprogramming factors include Oct3 / 4, Sox2, Soxl, Sox3, Soxl5, Soxl7, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbxl5, ERas, ECAT15-2, Tell, β-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glisl, and these reprogramming factors can be used alone or in combination of two or more of them.

[0060] If the cell modified by insertion of the induction cassette is for use in a human patient, it is preferred that the cell is an iPSC from that individual. The use of such autologous cells will eliminate the need for cell-receptor matching. Alternatively, commercially available iPSCs known to those skilled in the art can be used. Alternatively, the cell can be a tissue-specific stem cell, which can also be autologous or donated. Suitable cells include ectodermal stem cells, induced neural stem cells, and other tissue-specific stem cells.

[0061] In some embodiments of the methods of the present invention, it is preferred that the stem cells used are embryonic stem cells or stem cell lines. There are now many embryonic stem cell lines available commercially, such as WA01 (HI), WA09 (H9), KhES-1, KhES-2, and KhES-3. Stem cell lines derived without destroying the embryo are available. The present invention does not extend to any method involving the destruction of a human embryo.

[0062] As used herein, the term "targeted insertion" refers to insertion into a genomic safe harbor (GSH) site, which, as described elsewhere, is preferably specifically within the GSH sequence. Any suitable technique for inserting a polynucleotide into a specific sequence can be used, and some such techniques are described in the art. Suitable techniques include any method known to those skilled in the art that can introduce a break at the desired location and allow the vector to be recombined into the gap. Thus, the key first step in targeted site-specific genomic modification is to generate a double-strand DNA break (DSB) at the genomic locus to be modified. Different cellular repair mechanisms can be utilized to repair the DSB and introduce the desired sequence, and these different cellular repair mechanisms are the more error-prone non-homologous end joining repair (NHEJ); and homologous recombination repair (HR) mediated by a donor DNA template, which can be used to insert the induction cassette.

[0063] There are several techniques to allow site - specific generation of DSBs in the genome. Many of these techniques involve the use of custom endonucleases such as zinc - finger nucleases (ZFNs), transcription activator - like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats / CRISPR - associated protein (CRISPR / Cas9) systems (Gaj et al., 2013). Zinc - finger nucleases are artificial enzymes generated by fusing a zinc - finger DNA - binding domain to the nuclease domain of the restriction enzyme FokI. The latter has a non - specific cleavage domain that must dimerize to cut DNA. This means that two ZFN monomers are required to allow dimerization of the FokI domain and cut DNA. The DNA - binding domain can be designed to target any genomic sequence of interest, which can be a tandem array of Cys2His2 zinc fingers, each of which recognizes three consecutive nucleotides in the target sequence. The two binding sites are separated by 5 - 7 bp to allow optimal dimerization of the FokI domain. Thus, the enzyme is able to cut DNA at specific sites and increases target specificity by ensuring that two adjacent DNA - binding events must occur to achieve a double - strand break. Transcription activator - like effector nucleases or TALENs are dimeric transcription factor / nucleases. They are prepared by fusing a TAL effector DNA - binding domain to a DNA - cleavage domain (nuclease). Transcription activator - like effectors (TALENs) can be engineered to actually bind any desired DNA sequence, so when combined with a nuclease, DNA can be cut at a specific location. TAL effectors are proteins secreted by Xanthomonas, and their DNA - binding domain contains a repeated highly conserved 33 - 34 - amino - acid sequence with variable amino acids at positions 12 and 13. These two positions are highly variable and show a strong correlation with specific nucleotide recognition. This direct relationship between the amino - acid sequence and DNA recognition enables the engineering of a specific DNA - binding domain by selecting a combination of repeat segments containing appropriate residues at the two variable positions. Thus, TALENs are constructed from an array of 33 to 35 - amino - acid modules, each module targeting a single nucleotide. By selecting the module array, almost any sequence can be targeted. The nuclease used can be FokI or its derivatives.

[0064] Three CRISPR mechanisms have been identified, with type II being the most intensively studied. The CRISPR / Cas9 system (type II system) uses the Cas9 nuclease to generate double-strand breaks in DNA at sites determined by short guide RNAs. The CRISPR / Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements. CRISPR is a segment of prokaryotic DNA containing short repeats of base sequences. After each repeat is a short segment of "protospacer DNA" from a previous exposure to a foreign genetic element. CRISPR spacers use RNA interference to recognize and cut foreign genetic elements. The CRISPR immune response occurs in two steps: CRISPR-RNA (crRNA) biosynthesis and crRNA-guided interference. CrRNA molecules consist of variable sequences transcribed from protospacer DNA and CRISP repeats. Each crRNA molecule then hybridizes with a second RNA called trans-activating CRISPR RNA (tracrRNA), and the two together ultimately form a complex with the nuclease Cas9. The protospacer DNA-encoded portion of the crRNA guides Cas9 to cut complementary target DNA sequences if they are adjacent to a short sequence called the protospacer adjacent motif (PAM). In many other applications, this natural system has been engineered and used to introduce DSB breaks at specific sites in genomic DNA. In particular, the CRISP type II system from Streptococcus pyogenes can be used. Most simply, the CRISPR / Cas9 system contains two components that are delivered to cells to provide genome editing: the Cas9 nuclease itself and a small guide RNA (gRNA). The gRNA is a fusion of a custom-designed site-specific crRNA (targeted to the target sequence) and a standardized tracrRNA.

[0065] Once a DSB is prepared, a donor template that is homologous to the target locus is provided. The DSB can be repaired by the homology-directed repair (HDR) pathway that allows precise insertion. Derivatives of this system are also possible. Mutant forms of Cas9 can be utilized, such as Cas9D10A, which has only nickase activity. This means that it only cleaves one DNA strand and does not activate NHEJ. In contrast, when a homologous repair template is provided, DNA repair occurs only through the high-fidelity HDR pathway. Cas9D10A can be used in paired Cas9 complexes that are designed to generate adjacent DNA nicks complementary to adjacent regions on opposite strands of the target site with two sgRNAs, which can be particularly advantageous. Elements for creating double-stranded DNA breaks can be introduced into one or more vectors such as plasmids for expression in cells. Thus, any method that produces a specific, targeted double-stranded break in the genome to allow insertion of a nucleotide sequence / gene / inducible cassette can be used in the methods of the present invention. Preferably, the methods of the present invention are used to insert a gene / inducible cassette into any one or more of the ZFN, TALEN, and / or CRISPR / Cas9 systems or any derivatives thereof.

[0066] Once a DSB is prepared by any suitable method, the gene / inducible cassette for insertion can be provided in any suitable manner as described below. The gene / inducible cassette and associated genetic material form the donor DNA for repairing the DNA at the DSB and are inserted using standard cellular repair mechanisms / pathways. As described above, how the break is induced will alter which pathway is used to repair the damage. However, this is also within the knowledge of those skilled in the art.

[0067] As used in the present invention, the term "gene" means the basic physical unit of heredity, i.e., the linear sequence of nucleotides along a DNA segment that provides the coding instructions for the synthesis of RNA, which, when translated into protein, results in the expression of a genetic trait.

[0068] As used in the present invention, the term "nucleotide sequence" refers to a series of bases in a DNA segment that forms a gene as defined above.

[0069] As used herein, the term "transcription regulatory protein" means a protein that binds to DNA, preferably a sequence that specifically binds to a DNA site located in or near a promoter, which promotes the binding of the transcription machinery to the promoter, thereby promoting the transcription of a DNA sequence (transcription activator) or blocking the process (transcription repressor). Such entities are also referred to as transcription factors. The DNA sequence to which a transcription regulatory protein binds is called a transcription factor binding site or response element, which is present in the promoter of the regulated DNA sequence or in the vicinity of the promoter of the regulated DNA sequence. The response element is part of the present invention. A transcription activator protein binds to the response element and promotes gene expression. In the methods of the present invention, preferably these proteins are used to control the expression of the induction cassette. A transcription repressor protein binds to the response element and blocks gene expression. Transcription regulatory proteins can be activated or inactivated by many mechanisms, including the binding of substances, interactions with other transcription factors (such as homodimerization or heterodimerization) or co-regulatory proteins, phosphorylation, and / or methylation. Transcription regulatory factors can be controlled by activation or inactivation. If the transcription regulatory protein is a transcription activator protein, then preferably the transcription activator protein requires activation. This activation can be carried out in any suitable manner, but preferably, the transcription regulatory protein is activated by adding an exogenous substance to the stem cell. The supply of the exogenous substance to the stem cell can be controlled, so that the activation of the transcription regulatory protein can be controlled. Such transcription regulatory proteins are also referred to as inducible transcription regulatory proteins.

[0070] As used herein, the term "transcription factor" means a protein that binds to DNA, preferably a sequence that specifically binds to a DNA site located in or near a promoter, which promotes the binding of the transcription machinery to the promoter, thereby promoting the transcription of a DNA sequence (transcription activator) or blocking the process (transcription repressor). In the context of the present invention, the transcription factor is the genetic sequence required, preferably the DNA sequence transferred into the cell together with the induction cassette. The introduction of the induction cassette into the genome has the potential to change the phenotype of the cell by adding a genetic sequence that allows gene expression. The methods of the present invention provide for the controllable transcription of the genetic sequences of the transcription factor set within the induction cassette in a cell.

[0071] The master regulator can be one or more of the following: a transcription factor, a transcriptional regulator, a cytokine receptor, a signaling molecule, etc. The master regulator is an expressed gene that affects the lineage of the cell that expresses it. A network of master regulators may be required to determine cell lineage. As used herein, a master regulatory gene expressed at the beginning of a developmental lineage or cell type participates in the specification of that lineage by regulating multiple downstream genes either directly or through a cascade of gene expression changes. If the master regulator is expressed, it has the ability to re-determine the fate of cells destined to form other lineages. Transcription factors that can be used in the methods of the present invention include PU.1 (SEQ ID NO:2) (gene SPI1, SEQ ID NO:1), CEBPB (SEQ ID NO:3), RUNX1 (SEQ ID NO:4), IRF8 (SEQ ID NO:5), and SALL1 (SEQ ID NO:6).

[0072] As used in the present invention, the term "PU.1" (SEQ ID NO:2) refers to a transcription factor, also known as hematopoietic transcription factor PU.1, Spi-1 proto-oncogene, 31 kDa transforming protein, transcription factor PU.1, spleen focus-forming virus (SFFV) proviral integration oncogene Spi1, spleen focus-forming virus (SFFV) proviral integration oncogene or 31 kDa transforming protein, SFPI1, SPI-1, SPI-A, PU.1, or OF, wherein "SPI1" refers to the gene (SEQ ID NO:1) (Spi-1 proto-oncogene) that encodes an ETS-domain transcription factor that activates gene expression during myeloid and B lymphocyte development.

[0073] As used herein, the term "genomic safe harbor locus" refers to a genetic locus that permits the insertion of genetic material without deleterious effects on the cell and permits transcription of the inserted genetic material. Those skilled in the art can use these simplified criteria to identify suitable GSHs and / or use more formal criteria. Specific insertions within genomic safe harbor loci (GSHs) are preferred over random genomic integration because this is expected to be a safer modification of the genome and is less likely to result in unwanted side effects such as silencing of native gene expression or causing mutations that lead to cancer cell types. Thus, a genomic safe harbor locus is a locus within the genome where a gene or other genetic material can be inserted without any deleterious effects on the cell or on the inserted genetic material. Most beneficially, GSH loci where the inserted gene sequence is expressed are not perturbed by any read-through expression from neighboring genes and expression of the induction cassette, thus minimizing interference with the endogenous transcriptional program. More formal criteria have been proposed that assist in determining whether a particular locus is a GSH locus (Pellenz et al., 2019). These criteria include loci that are: (i) >300 kb from any cancer-related gene on a list of all oncogenes, (ii) >300 kb from any miRNA / other functional small RNA, (iii) >50 kb from any 5' gene end, (iv) >50 kb from any replication origin, (v) >50 kb from any ultra-conserved element, (vi) loci with low transcriptional activity (no mRNA ±25 kb), (vii) loci not in copy number variable regions, (viii) loci in open chromatin (DHS signal ±1 kb) and (ix) unique loci (1 copy in the human genome). It may not be necessary to meet all of these proposed criteria as identified GSHs do not meet all of these criteria. Preferably, a suitable GSH can meet at least 3, 4, 5, 6, 7 or 8 of these criteria, most preferably all 9 criteria.

[0074] In the method of the invention, the insertions occur at different GSHs. At least two GSHs are required. The first GSH is modified by insertion of a transcriptional regulatory protein. The second GSH is modified by insertion of an induction cassette that contains a coding sequence operably linked to an inducible promoter. Other genetic material may also be inserted with one or both of these elements. Preferably, the genetic sequence operably linked to the inducible promoter within the induction cassette is a DNA sequence. Preferably, the genetic sequence of the induction cassette encodes an RNA molecule and is thus capable of being transcribed. Transcription is controlled using an inducible promoter. The RNA molecule can be of any sequence but is preferably an mRNA, shRNA or gRNA encoding a protein.

[0075] The first GSH can be any suitable GSH locus. Optionally, the first GSH is a GSH with an endogenous promoter that is constitutively expressed, which will result in constitutive expression of the inserted transcriptional regulatory protein. A suitable GSH is the hROSA26 locus of human cells. In another embodiment of the present invention, the inserted transcriptional regulatory protein operably linked to the promoter is a constitutive promoter. The constitutive promoter can be used, for example, in combination with the insertion at the hROSA26 locus.

[0076] As used in the present invention, the term "inducible promoter" means a nucleotide sequence that initiates and regulates the transcription of a polynucleotide. An "inducible promoter" is a nucleotide sequence in which the expression of the genetic sequence operably linked to the promoter is controlled by an analyte, cofactor, regulatory protein, etc. In one embodiment of the method of the present invention, the control is affected by a transcriptional regulatory protein. The term "promoter" or "control element" includes the full-length promoter region and functional (e.g., controlling transcription or translation) segments of these regions. Preferably, the gene encoding the transcriptional regulatory protein is operably linked to a constitutive promoter. Alternatively, the first GSH can be selected such that it already has a constitutive promoter that can drive the expression of the transcriptional regulatory protein gene and any associated genetic material. The constitutive promoter ensures continuous and high-level gene expression. Commonly used constitutive promoters include the human β-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-la (EFla), phosphoglycerate kinase (PGK), and ubiquitin C (UbC). The CAG promoter is a strong synthetic promoter that is often used to drive high-level gene expression.

[0077] As used in the present invention, the term "culturing" means the growth of microorganisms such as bacteria and yeast, or human, plant, or animal cells under suitable conditions that ensure growth, and such suitable conditions are within the knowledge of those skilled in the art.

[0078] As used in the present invention, the term "growth factor" means a signaling molecule that controls cell activity in an autocrine, paracrine, or endocrine manner. As used herein, in the context of the present invention, the term "growth factor" can be used interchangeably with "cytokine". Growth factors or cytokines are produced by different cell types of an organism and exert their biological functions by binding to specific receptors and activating related downstream signaling pathways, which in turn regulate gene transcription in the nucleus and ultimately stimulate a biological response, which includes regulatory cell processes such as cell division, cell survival, cell differentiation, adhesion, and migration.

[0079] As used in the present invention, the term "small molecule" means a bioactive molecule, natural or man-made, which is capable of diffusing through the cell membrane and of modulating signaling pathways. Preferred small molecules for use in the present invention can inhibit phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase 3, such as LY294002 and CHIR99021, respectively.

[0080] As used in the present invention, the term "recapitulate signaling" means to mimic, imitate or emulate the function of secreted molecules such as growth factors and / or chemokines, which affect cells in their natural environment and are thereby capable of generating microglia through these actions.

[0081] As used in the present invention, the term "mimic signaling" means to mimic, imitate, emulate or recapitulate the function of secreted molecules such as growth factors and / or chemokines, which affect cells in their natural environment and are thereby capable of generating microglia through these actions.

[0082] As used in the present invention, the term "embryonic development of microglia" means the progressive conversion of pluripotent stem cells into mature microglia, starting from the pre-implantation blastocyst-stage embryo until a fully established and self-maintaining microglial population, as a result of microglial differentiation during human embryonic, fetal and postnatal development.

[0083] As used in the present invention, the term "adult microglial proliferation" means any process of cell division leading to mature microglia.

[0084] As used in the present invention, the term "adult microglial differentiation" means the differentiation of cells in the microglial progenitor state into adult microglial cell types, which introduce the typical features of microglia in the homeostatic / quiescent state.

[0085] As used in the present invention, the term "adult microglial polarization" means the response of mature microglia to extracellular stimuli provided by the extracellular environment, which are signals from damaged neurons, glial cells or exposure to plasma proteins due to blood-brain barrier dysfunction, respectively. This microglial response includes the movement of microglia towards the site of injury and can have a neuroprotective or toxic effect.

[0086] In addition, in one embodiment of the method of the present invention, the at least one growth factor or small molecule is selected from activin A (SEQ ID NO:7), BMP4 (SEQ ID NO:8), FGF (SEQ ID NO:9), VEGF-A (SEQ ID NO:10), LY294002, CHIR99021, SCF (SEQ ID NO:11), IL-3 (SEQ ID NO:12), IL-6 (SEQ ID NO:13), CSF1 (SEQ ID NO:14), IL-34 (SEQ ID NO:15), CSF2 (SEQ ID NO:16), CD200 (SEQ ID NO:17), CX3CL1 (SEQ ID NO:18), TGFβ1 (SEQ ID NO:19), and IDE1.

[0087] As used herein, activin A (SEQ ID NO:7) refers to activin β-A chain, EDF, erythroid differentiation protein, FRP, FSH releasing protein, INHBA, inhibin β-A chain, inhibin β-1. The protein encoded by this gene is a member of the transforming growth factor β (TGF-β) protein family produced by pluripotent stem cells, endoderm, and mesoderm.

[0088] As used herein, BMP4 (SEQ ID NO:8) refers to bone morphogenetic protein 4, also known as ZYME, BMP2B, or BMP2B1. The protein encoded by this gene is a member of the bone morphogenetic protein family, which is part of the transforming growth factor-β superfamily.

[0089] As used herein, FGF (SEQ ID NO:9) refers to fibroblast growth factor. The protein encoded by this gene is a member of the family of cell signaling proteins as described in Hui et al., 2018.

[0090] As used herein, VEGF-A (SEQ ID NO:10) refers to vascular endothelial growth factor A, also known as VPF, VEGF, or MVCD1. The protein encoded by this gene is a member of the PDGF / VEGF growth factor family and is a heparin-binding protein. This growth factor induces the proliferation and migration of vascular endothelial cells and is essential for both physiological and pathological angiogenesis.

[0091] As used herein, LY294002 refers to a potent cell-permeable inhibitor of phosphatidylinositol 3-kinase (PI3K) that acts on the ATP-binding site of the enzyme (Vlahos et al., 1994). Its chemical structure is as follows:

[0092]

[0093] As used in the present invention, CHIR99021 refers to an aminopyrimidine derivative that is a highly effective inhibitor of glycogen synthase kinase 3, which inhibits GSK3β (IC 50 = 6.7 nM) and GSK3α (IC 50 = 10 nM) and acts as a WNT activator. Its chemical structure is as follows:

[0094]

[0095] As used in the present invention, SCF (SEQ ID NO:11) refers to stem cell factor, also known as kit ligand, mast cell growth factor or Steel factor. The protein encoded by this gene is an early-acting cytokine that plays a key role in the regulation of embryonic and adult hematopoiesis.

[0096] As used in the present invention, IL-3 (SEQ ID NO:12) refers to interleukin-3, MCGF (mast cell growth factor), multi-CSF, HCGF, P-cell stimulating factor, MGC79398 or MGC79399. The protein encoded by this gene is a growth-promoting cytokine.

[0097] As used in the present invention, IL-6 (SEQ ID NO:13) refers to interleukin 6, also known as B-cell stimulating factor 2, CTL differentiation factor, hybridoma growth factor, interferon β-2, interleukin-6, IFN-β-2, IFNB2, BSF-2, CDF, interferon, β2, B cell differentiation factor, interferon, β2, interleukin BSF-2, BSF2, HGF or HSF. The protein encoded by this gene is a cytokine that plays a role in inflammation and B cell maturation.

[0098] As used in the present invention, CSF1 (SEQ ID NO:14) refers to colony-stimulating factor 1, also known as colony-stimulating factor 1 (macrophage), macrophage colony-stimulating factor 1, macrophage colony-stimulating factor 1, Lanimostim, CSF-1, MCSF, M-CSF, and the protein encoded by this gene is a cytokine that controls the production, differentiation and function of macrophages.

[0099] As used in the present invention, IL-34 (SEQ ID NO:15) refers to interleukin 34, also known as C16 or f77. The protein encoded by this gene is a cytokine that promotes the differentiation and viability of monocytes and macrophages through the colony-stimulating factor-1 receptor.

[0100] As used in the present invention, CSF2 (SEQ ID NO:16) refers to colony-stimulating factor 2, also known as Sarcramostim, colony-stimulating factor 2 (granulocyte-macrophage), granulocyte-macrophage colony-stimulating factor, Molgramostin, molgramostim, GMCSF, CSF, granulocyte macrophage-colony stimulating factor, granulocyte-macrophage colony-stimulating factor, colony-stimulating factor, GM-CSF. The protein encoded by this gene is a cytokine that controls the production, differentiation, and function of granulocytes and macrophages.

[0101] As used in the present invention, CD200 (SEQ ID NO:17) refers to the CD200 gene, also known as CD200 molecule, CD200 antigen, antigen recognized by monoclonal antibody MRC OX-2, OX-2 membrane glycoprotein, MOX1, MOX2, OX-2, or MRC. The protein encoded by this gene is a type I membrane glycoprotein containing two extracellular immunoglobulin domains, a transmembrane domain, and a cytoplasmic domain.

[0102] As used in the present invention, CX3CL1 (SEQ ID NO:18) refers to the CX3CL1 gene, also known as C-X3-C motif chemokine ligand 1, small inducible cytokine subfamily D (Cys-X3-Cys), member 1 (chemokine fractalkine, neurotactin), chemokine (C-X3-C motif) ligand 1, CX3C membrane-anchored chemokine, small inducible cytokine D1, C-X3-C motif chemokine 1, neurotactin, chemokine fractalkine, or SCYD1, NTT, small inducible cytokine subfamily D (Cys-X3-Cys), member-1, C3Xkine, ABCD-3, CXC3C, CXC3, NTN, or FKN. The protein encoded by this gene belongs to the CX3C subgroup of chemokines, which is characterized by the number of amino acids located between conserved cysteine residues.

[0103] As used in the present invention, TGFβ1 (SEQ ID NO:19) refers to transforming growth factor β1, also known as transforming growth factor β-1 proprotein, precursor transforming growth factor β-1, TGFB, transforming growth factor β1, transforming growth factor β-1, latency-associated peptide, Camauri-Engelmann disease, TGF-β-1, IBDIMDE, TGFβ, DPD1, CED, or LAP. The protein encoded by this gene is a secreted ligand of the TGF-β (transforming growth factor-β) protein superfamily.

[0104] In another embodiment of the method of the present invention, the at least one growth factor is CSF1 (SEQ ID NO: 14) or IL-34 (SEQ ID NO: 15). In yet another embodiment of the method of the present invention, the at least one growth factor is CSF1 (SEQ ID NO: 14). In still another embodiment of the method of the present invention, the at least one growth factor is IL-34 (SEQ ID NO: 15).

[0105] In another embodiment of the method of the present invention, the at least one small molecule is CHIR99021, LY294002 or IDE1.

[0106] As used in the present invention, LY294002 refers to a potent cell-permeable inhibitor of phosphatidylinositol 3-kinase (PI3K) that acts on the ATP-binding site of the enzyme (Vlahos et al., 1994). Its chemical structure is as follows:

[0107]

[0108] As used in the present invention, CHIR99021 refers to an aminopyrimidine derivative that is a potent inhibitor of glycogen synthase kinase 3, which inhibits GSK3β (IC 50 = 6.7 nM) and GSK3α (IC 50 = 10 nM) and acts as a WNT activator. Its chemical structure is as follows:

[0109]

[0110] As used in the present invention, IDE1 refers to an inducer of definitive endoderm; a small molecule that activates the TGF-β pathway and can be used as a substitute for the growth factor TGF-β. Its chemical structure is as follows:

[0111]

[0112] In another embodiment of the method of the present invention, the first and second genomic safe harbor sites are different.

[0113] In yet another embodiment of the method of the present invention, the method further comprises the insertion and expression of the coding sequence (SEQ ID NO: 3) of the gene for the transcription factor CEBPB.

[0114] As used in the present invention, CEBPB (SEQ ID NO:3) refers to CCAAT / enhancer binding protein β, also known as CCAAT / enhancer binding protein β, CCAAT / enhancer binding protein (C / EBP), β, interleukin 6-dependent DNA binding protein, CCAAT / enhancer binding protein β, nuclear factor for interleukin 6, transcription factor 5, nuclear factor NF-IL6, TCF5, liver-enriched transcriptional activator protein, CCAAT / enhancer binding protein β, liver-enriched inhibitory protein, transcription factor C / EBPβ, liver activator protein, C / EBP-β, C / EBPβ, IL6DBP, NF-IL6, TCF-5, LAP or LIP. This intronless gene encodes a transcription factor containing a basic leucine zipper (bZIP) domain.

[0115] In another embodiment of the method of the present invention, the method further comprises the insertion and expression of the coding sequence (SEQ ID NO:4) of the gene of transcription factor RUNX1.

[0116] As used in the present invention, RUNX1 (SEQ ID NO:4) refers to Runt-related transcription factor 1, Runt-related transcription factor 1, polyomavirus enhancer-binding protein 2 alpha B subunit, SL3 / AKV core-binding factor alpha B subunit, SL3-3 enhancer factor 1 alpha B subunit, acute myeloid leukemia 1 protein, oncogene AML-1, PEBP2-alpha B, PEA2-alpha B, CBFA2, AML1, core-binding factor Runt domain alpha subunit 2 core-binding factor subunit alpha-2, AML1-EVI-1 fusion protein, acute myeloid leukemia, Aml1 oncogene, CBF-alpha-2, AML1-EVI-1, PEBP2 alpha, CBF2 alpha, PEBP2aB, AMLCR1 or EVI-1. The protein encoded by this gene represents the alpha subunit of CBF and is thought to be involved in the development of normal hematopoiesis.

[0117] In another embodiment of the method of the present invention, the method further comprises the insertion and expression of the coding sequence (SEQ ID NO:5) of the gene of transcription factor IRF8.

[0118] As used in the present invention, IRF8 (SEQ ID NO:5) refers to interferon regulatory factor 8, also known as interferon consensus sequence binding protein 1, H-ICSBP, ICSBP1, ICSBP, IRF-8, interferon consensus sequence binding protein, IMD32A, IMD32B or interferon consensus sequence binding protein (ICSBP). It is a transcription factor of the interferon (IFN) regulatory factor (IRF) family.

[0119] In another embodiment of the method of the present invention, the method further comprises the insertion and expression of the coding sequence (SEQ ID NO:6) of the gene of the transcription factor SALL1.

[0120] As used in the present invention, SALL1 (SEQ ID NO:6) refers to spalt-like transcription factor 1, also known as zinc finger protein spalt-1, zinc finger protein SALL1, zinc finger protein 794, Sal-like protein 1, ZNF794, Sal-1, epididymal secretory protein Li89, spalt-like transcription factor 1, Sal (Drosophila)-like 1, Sal-like 1 (Drosophila), HEL-S-89, HSAL1, HSal1, SAL1 or TBS. The protein encoded by this gene is a zinc finger transcriptional repressor and can be part of the NuRD histone deacetylase complex (HDAC).

[0121] In another embodiment of the method of the present invention, the transcriptional regulatory protein is reverse tetracycline transactivator (rtTA) (SEQ ID NO:20), the activity of which is controlled by doxycycline or tetracycline.

[0122] As used in the present invention, the term "reverse tetracycline transactivator (rtTA)" refers to a transcriptional activator protein induced by tetracycline or its derivatives. Tetracycline-controlled transcriptional activation is a method of inducible gene expression, in which transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (such as the more stable doxycycline). In this system, the transcriptional activator protein can be a tetracycline-responsive transcriptional activator protein (rtTa) or its derivatives. The transcriptional regulatory protein of the present invention can be rtTA. The rtTA protein is capable of binding to DNA at specific TetO operator sequences. Placing several repeats of such TetO sequences upstream of a minimal promoter (such as the CMV promoter), together they form a tetracycline response element (TRE) (SEQ ID NO:21). Depending on whether the addition of tetracycline or its derivative activates (Tet-on) or inactivates (Tet-off) the rtTA protein, the system is divided into two forms. In one embodiment of the method of the present invention, the Tet-ON system in which doxycycline activates the rtTA protein can also be used.

[0123] The Tet-On system consists of two components; (1) a tetracycline-responsive transcriptional activator protein (rtTA) that is constitutively expressed and an rtTA-sensitive inducible promoter (tetracycline response element, TRE). This system can be bound by tetracycline or a more stable derivative of tetracycline, including doxycycline (dox), resulting in the activation of rtTA, its binding to the TRE sequence, and the induction of the expression of the gene controlled by TRE. The use of such a system in the methods of the present invention may be preferred. Thus, the transcriptional regulatory protein of the methods of the present invention can be the tetracycline-responsive transcriptional activator protein (rtTA), which can be activated or inactivated by an exogenously provided antibiotic tetracycline or one of its derivatives. If the transcriptional regulatory protein is rtTA, then the inducible promoter inserted into the second GSH site includes the tetracycline response element (TRE). The exogenously provided substance can be an antibiotic tetracycline or one of its derivatives, such as doxycycline, preferably tetracycline or doxycycline.

[0124] Variant and modified rtTA proteins can be used in the methods of the present invention. They can include Tet-On Advanced Transactivator (also known as rtTA2S-M2) and Tet-On 3G (also known as rtTA-V16, derived from rtTA2S-S2).

[0125] In another embodiment of the methods of the present invention, the inducible promoter includes the tetracycline response element (TRE) (SEQ ID NO:21).

[0126] As used in the present invention, the term "tetracycline response element (TRE)" means 7 repeats of a 19bp bacterial TetO sequence separated by spacer sequences and having a minimal promoter. Since the minimal promoter can be any suitable promoter, variants and modifications of the TRE sequence are possible. Preferably, in the absence of rtTA binding, the minimal promoter shows no expression level or shows a minimal expression level. Thus, the inducible promoter inserted into the second GSH can contain TRE. The basic genetic principle of the present invention is also described in Figure 2 which shows different GSH sites (hROSA26 and AAVS1) and the integrated rtTA (SEQ ID NO:20) and TRE (SEQ ID NO:21).

[0127] In another embodiment of the method of the present invention, the first and the second genomic safe harbor sites are selected from the hROSA26 locus (SEQ ID NO:22), the AAVS1 locus (SEQ ID NO:23), the CLYBL gene (SEQ ID NO:24), the CCR5 gene (SEQ ID NO:25), the HPRT gene (SEQ ID NO:26), or genes having locus ID 325 on chromosome 8 (SEQ ID NO:27), locus ID 227 on chromosome 1 (SEQ ID NO:28), locus ID 229 on chromosome 2 (SEQ ID NO:29), locus ID 255 on chromosome 5 (SEQ ID NO:30), locus ID 259 on chromosome 14 (SEQ ID NO:31), locus ID 263 on chromosome X (ID NO:32), locus ID 303 on chromosome 2 (SEQ ID NO:33), locus ID 231 on chromosome 4 (SEQ ID NO:34), locus ID 315 on chromosome 5 (SEQ ID NO:35), locus ID 307 on chromosome 16 (SEQ ID NO:36), locus ID 285 on chromosome 6 (SEQ ID NO:37), locus ID 233 on chromosome 6 (SEQ ID NO:38), locus ID 311 on chromosome 134 (SEQ ID NO:39), locus ID 301 on chromosome 7 (SEQ ID NO:40), locus ID 293 on chromosome 8 (SEQ ID NO:41), locus ID 319 on chromosome 11 (SEQ ID NO:42), locus ID 329 on chromosome 12 (SEQ ID NO:43), locus ID 313 on chromosome X (SEQ ID NO:44). Preferably, in another embodiment of the method of the present invention, the first and the second genomic safe harbor sites are selected from the hROSA26 locus (SEQ ID NO:22), the AAVS1 locus (SEQ ID NO:23), the CLYBL gene (SEQ ID NO:24), the CCR5 gene (SEQ ID NO:25), the HPRT gene (SEQ ID NO:26). More preferably, the first and the second genomic safe harbor sites are selected from the hROSA26 locus (SEQ ID NO:22) and the AAVS1 locus (SEQ ID NO:23).

[0128] Other sites can be identified by looking for sites where the virus integrates naturally without disrupting native gene expression. For the method of the present invention, several GSH sites can be used, which will be described in more detail below.

[0129] The adeno-associated virus integration site 1 locus (AAVS1) (SEQ ID NO:23) is located within the protein phosphatase 1, regulatory subunit 12C (PPP1R12C) gene on human chromosome 19, and it is expressed uniformly and ubiquitously in human tissues. This locus is identified as a possible GSH as it serves as the specific integration locus for AAV serotype 2. Since AAVS1 contains an open chromatin structure and natural chromatin insulators that confer resistance to silencing on the induction cassette, it has been shown to be a favorable environment for transcription. There are no known adverse effects on cells resulting from disruption of the PPP1R12C gene. Additionally, the induction cassette inserted at this locus maintains transcriptional activity in many different cell types. Therefore, AAVS1 is considered a GSH and has been widely used for targeted transgenesis in the human genome.

[0130] The hROSA26 locus (SEQ ID NO:22) was identified based on similarity to the sequence of a GSH from mouse (ROSA26 - reverse splice acceptor site #26). Although the orthologous locus has been identified in humans, this locus is not commonly used for induction cassette insertion. The inventors of the present invention used a targeting system specific to the hROSA26 locus and were thereby able to insert genetic material into this locus. The hROSA26 locus (SEQ ID NO:22) is on chromosome 3 (3p25.3) and can be found in the Ensembl database (GenBank: CR624523). The precise genomic coordinates of the integration site are 3:9396280 - 93963030:Ensembl. The integration site is within the open reading frame (ORF) of the THUMPD3 long non - coding RNA (reverse strand). Since the hROSA26 locus has an endogenous promoter, the inserted genetic material can utilize this endogenous promoter or, alternatively, be inserted operably linked to a promoter.

[0131] Intron 2 of the citrate lyase beta - like (CLYBL) gene (SEQ ID NO:24) on the long arm of chromosome 13 was identified as a suitable GSH as it is one of the integration hotspots recognized by the phage - derived phiC31 integrase. Studies have demonstrated that induction cassettes randomly inserted at this locus are stable and expressed. It has been shown that insertion of an induction cassette at this GSH does not interfere with local gene expression (Cerbibi et al., 2015). Therefore, CLYBL provides a GSH that can be used in the methods of the present invention.

[0132] CCR5 (SEQ ID NO:25), which is located on chromosome 3 (position 3p21.31), is a gene encoding the major co-receptor for HIV-1. The interest in using this locus as a GSH comes from a null mutation in this gene, which seems to have no side effects but confers resistance to HIV-1 infection. Zinc finger nucleases targeting the third exon have been developed, allowing the insertion of genetic material at this locus. Given that the natural function of CCR5 has not been elucidated, this locus remains a putative GSH for the methods of the present invention. The hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene encodes a transferase that plays a key role in the production of purine nucleotides via the purine salvage pathway. It has been proposed as a GSH locus. Insertion at this locus may be more suitable for mature cell types, such as modification for gene therapy. GSHs have been identified in other organisms and include the ROSA26, HRPT, and Hippll (Hll) loci in mice.

[0133] The mammalian genome may include GSH loci based on pseudo attP sites. For these loci, the hiC31 integrase, a Streptomyces phage-derived recombinase, has been developed as a non-viral insertion tool because of its ability to integrate plasmids carrying an induction cassette with an attB site into the pseudo attP sites. GSHs also exist in the genomes of plants, and the modification of plant cells can be used in the methods of the present invention. GSHs have been identified in the rice genome (Cantos et al., 2014).

[0134] The following SHS loci can be used in any method of the present invention. They were published by Pellenz et al. in 2019 and meet 5 of the 9 criteria listed above: locus ID 325 on chromosome 8: 68,720,172-68,720,191 (SEQ ID NO:27); locus ID 227 on chromosome 1: 231,999,396-231,999,415 (SEQ ID NO:28); locus ID 229 on chromosome 2: 45,708,354-45,708,373 (SEQ ID NO:29); locus ID 255 on chromosome 5: 19,069,307-19,069,326 (SEQ ID NO:30); locus ID 259 on chromosome 14: 92,099,558-92,099,577 (SEQ ID NO:31); locus ID 263 on chromosome X: 12,590,812-12,590,831 (SEQ ID NO:32); locus ID 303 on chromosome 2: 77,263,930-77,263,949 (SEQ ID NO:33); locus ID 317 on chromosome 2: 77,263,930-77,263,949 (SEQ ID NO:60); locus ID 231 on chromosome 4: 58,976,613-58,976,632 (SEQ ID NO:34); locus ID 315 on chromosome 5: 7,577,728-7,577,747 (SEQ ID NO:35); locus ID 307 on chromosome 16: 19,323,777-19,323,796 (SEQ ID NO:36); locus ID 285 on chromosome 6: 89,574,320-89,574,339 (SEQ ID NO:37); locus ID 233 on chromosome 6: 114,713,905-114,713,924 (SEQ ID NO:38); locus ID 311 on chromosome 6: 134,385,946-134,385,965 (SEQ ID NO:39); locus ID 301 on chromosome 7: 113,327,685-113,327,704 (SEQ ID NO:40); locus ID 293 on chromosome 8: 40,727,927-40,727,946 (SEQ ID NO:41); locus ID 319 on chromosome 11: 32,680,546-32,680,565 (SEQ ID NO:42); locus ID 329 on chromosome 12: 126,152,581-126,152,600 (SEQ ID NO:43);and locus ID 313: 16,059,732-16,059,751 (SEQ ID NO:44) on chromosome X;

[0135] In another embodiment of the method of the present invention, the stem cells are pluripotent stem cells, induced pluripotent stem cells (iPSCs), neural progenitor cells, hematopoietic stem cells or embryonic stem cells (ESCs).

[0136] In the present invention, the term "pluripotent stem cell" is used as defined above.

[0137] As used in the present invention, the term "neural progenitor cell" refers to a pluripotent cell state between pluripotent stem cells and mature somatic cells. This cell state is generally determined to become specialized cell types, such as neurons, oligodendrocytes and astrocytes.

[0138] In the present invention, the term "induced pluripotent stem cell (iPSC)" is used as defined above.

[0139] As used in the present invention, the term "hematopoietic stem cell" refers to a hematopoietic stem cell. This particular type of pluripotent stem cell is capable of forming any type of blood cell, but has lost the ability to form other cell types.

[0140] In the present invention, the term "embryonic stem cell (ESC)" is used as defined above.

[0141] In another embodiment of the method of the present invention, the stem cells are human or mouse stem cells.

[0142] As used in the present invention, the term "human or mouse stem cell" refers to a cell from a human or a mouse. However, the stem cells used in the method of the present invention can be any human or animal cell. Preferably mammalian cells, such as cells from rodents such as mice and rats; marsupials, such as kangaroos and wombats; non-human primates, such as pygmy chimpanzees, chimpanzees, lemurs, gibbons and apes; camelids, such as camels and llamas; livestock, such as horses, pigs, cows, buffalo, bison, goats, sheep, deer, reindeer, donkeys, banteng, yaks, chickens, ducks and turkeys; domestic animals, such as cats, dogs, rabbits and guinea pigs. Preferably, the cells are human cells. In some aspects, the cells are preferably from livestock. Once the insertion of the genetic material into the GSH locus is complete, the type of cell used in the method of the present invention will depend on the application of the cell.

[0143] The present invention also relates to microglial cells obtained by any method according to the present invention, preferably wherein said microglial cells express at least one microglial cell surface protein selected from ITGAM (CD11B) (SEQ ID NO: 45), ITGAX (CD11C) (SEQ ID NO: 46), CD14 (SEQ ID NO: 47), CD16 (SEQ ID NO: 48), ENTPD1 (CD39) (SEQ ID NO: 49), PTPRC (CD45) (SEQ ID NO: 50), CD68 (SEQ ID NO: 51), CSF1R (CD115) (SEQ ID NO: 52), CD163 (SEQ ID NO: 53), CX3CR1 (SEQ ID NO: 54), TREM2 (SEQ ID NO: 55), P2RY12 (SEQ ID NO: 56), TMEM119 (SEQ ID NO: 57), and HLA-DR (SEQ ID NO: 58).

[0144] Thus, microglial cells are further defined by expressing at least one of the following surface proteins: ITGAM (CD11B) (SEQ ID NO: 45), ITGAX (CD11C) (SEQ ID NO: 46), CD14 (SEQ ID NO: 47), CD16 (SEQ ID NO: 48), ENTPD1 (CD39) (SEQ ID NO: 49), PTPRC (CD45) (SEQ ID NO: 50), CD68 (SEQ ID NO: 51), CSF1R (CD115) (SEQ ID NO: 52), CD163 (SEQ ID NO: 53), CX3CR1 (SEQ ID NO: 54), TREM2 (SEQ ID NO: 55), P2RY12 (SEQ ID NO: 56), TMEM119 (SEQ ID NO: 57), and HLA-DR (SEQ ID NO: 58). These proteins are defined as follows.

[0145] As used in the present invention, ITGAM (CD11B) refers to integrin subunit alpha M, a gene encoding the integrin alpha M chain. Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain. The protein sequence of ITGAM (CD11B) is given in SEQ ID NO: 45.

[0146] As used in the present invention, ITGAX (CD11C) refers to integrin subunit alpha X, and the gene encodes the integrin alpha X chain protein. The protein sequence of ITGAX (CD11C) is given in SEQ ID NO: 46.

[0147] As used in the present invention, CD14 refers to the monocyte differentiation antigen CD14, and the protein encoded by this gene is a surface antigen preferably expressed on monocytes / macrophages. The protein sequence of CD14 is given in SEQ ID NO:47.

[0148] As used in the present invention, CD16 refers to the FCGR3A Fc fragment of IgG receptor IIIa, and the gene encodes a receptor for the Fc portion of immunoglobulin G, which is involved in the removal of antigen-antibody complexes from the circulation and other antibody-dependent responses. The protein sequence of CD16 is given in SEQ ID NO:48.

[0149] As used in the present invention, ENTPD1 (CD39) refers to ectonucleoside triphosphate diphosphohydrolase 1, and the protein encoded by this gene is a plasma membrane protein that hydrolyzes extracellular ATP and ADP to AMP. The protein sequence of ENTPD1 (CD39) is given in SEQ ID NO:49.

[0150] As used in the present invention, PTPRC (CD45) refers to protein tyrosine phosphatase receptor type C, and the protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family. The protein sequence of PTPRC (CD45) is given in SEQ ID NO:50.

[0151] As used in the present invention, CD68 refers to the CD68 antigen, and the gene encodes an 110-kD transmembrane glycoprotein highly expressed by human monocytes and tissue macrophages. The protein sequence of CD68 is given in SEQ ID NO:51.

[0152] As used in the present invention, CSF1R (CD115) refers to colony-stimulating factor 1 receptor, and the protein encoded by this gene is a receptor for colony-stimulating factor 1, which is a cytokine that controls the production, differentiation, and function of macrophages. The protein sequence of CSF1R (CD115) is given in SEQ ID NO:52.

[0153] As used in the present invention, CD163 refers to the CD163 antigen, and the protein encoded by this gene is a member of the scavenger receptor cysteine-rich (SRCR) superfamily and is expressed only in monocytes and macrophages. The protein sequence of CD163 is given in SEQ ID NO:53.

[0154] As used in the present invention, CX3CR1 refers to C-X3-C motif chemokine receptor 1, and the protein encoded by this gene is the receptor for the chemokine fractalkine. The protein sequence of CX3CR1 is given in SEQ ID NO:54. Fractalkine is a transmembrane protein and chemokine involved in the adhesion and migration of leukocytes.

[0155] As used in the present invention, TREM2 refers to triggering receptor expressed on myeloid cells 2, and the gene encodes a membrane protein that forms a receptor signaling complex with TYRO protein tyrosine kinase-binding protein. The protein sequence of TREM2 is given in SEQID NO:55.

[0156] As used in the present invention, P2RY12 refers to purinergic receptor P2Y12, and the product of this gene belongs to the G-protein coupled receptor family. The protein sequence of P2RY12 is given in SEQ ID NO:56.

[0157] As used in the present invention, TMEM119 refers to transmembrane protein 119, which is a gene encoding a protein. Its related pathway is microglial activation during neuroinflammation. The protein sequence of TMEM119 is given in SEQ ID NO:57.

[0158] As used in the present invention, HLA-DR refers to major histocompatibility complex, class II, DRα and β, and both HLA-DRA and HLA-DRB1 are paralogs of the HLA class II α chain. The protein sequence of HLA-DR is given in SEQ ID NO:58.

[0159] In a further embodiment, the present invention also includes microglia according to the present invention for use in therapy.

[0160] As used in the present invention, the term "treatment" refers to any form of treatment of a disease or an undesirable health state in an organism, animal or human. It can also include gene therapy. Gene therapy can be defined as the artificial insertion of foreign DNA into the cell nucleus for therapeutic purposes. Such a definition includes providing one or more genes to a cell in order to provide a wild-type form of a faulty gene, adding genes that interfere with the expression of a target gene (which may be defective), providing suicide genes (such as enzymes that convert the harmless prodrug ganciclovir (GCV) into a cytotoxic drug - herpes simplex virus thymidine kinase (HSV-tk) and cytosine deaminase (CD) enzymes), providing DNA vaccines for immunotherapy or cancer therapy (including adoptive cell immunotherapy), and providing any other genes to a cell for therapeutic purposes. In addition, mature microglial cells can be directly used for transplantation into a human or an animal. Optionally, microglial cells can form test materials for research, including the effects of drugs on gene expression and the interaction of drugs with specific genes. Microglial cells for research can involve the use of an induction cassette with a gene sequence of unknown function to study the controllable expression of the gene sequence. In addition, microglial cells can be used to produce large amounts of required materials, such as growth factors or cytokines.

[0161] Furthermore, in one embodiment, the present invention also relates to the use of such microglial cells according to the present invention for in vitro diagnosis of diseases. Preferably, the diseases are selected from central nervous system diseases, preferably neurodegenerative diseases; more preferably Alzheimer's disease, Parkinson's disease, frontotemporal dementia or amyotrophic lateral sclerosis; neuroinflammatory or autoimmune diseases, preferably multiple sclerosis, autoimmune antibody-mediated encephalitis or infectious diseases, neurovascular diseases; preferably stroke, vasculitis; traumatic brain injury and cancer.

[0162] Furthermore, the present invention relates to the use of such microglial cells according to the present invention for in vitro co-culture with brain organoids.

[0163] As used in the present invention, the term "organoid" means an in vitro 3D-organ model derived mainly from (stem) cells, and in combination with microglial cells produced according to the present invention represents a powerful tool for medical diagnosis for studying the participation and interaction of microglial cells with other cells of the brain.

[0164] ***

[0165] Note that, unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an", and "the" include plural referents. Thus, for example, a reference to "a reagent" includes one or more such reagents, and a reference to "the method" includes references to equivalent steps and methods known to those of ordinary skill in the art, which may modify or replace the methods described herein.

[0166] Unless otherwise specified, the term "at least" before a series of elements should be understood to refer to each element in the series. If not specifically defined differently, the term "at least one" refers to one or more, such as two, three, four, five, six, seven, eight, nine, ten or more. Those skilled in the art will recognize or be able to use many equivalents of the specific embodiments of the invention described herein without more than routine experimentation. These equivalents are also included in the invention.

[0167] As used herein, the term "and / or" includes the meanings of "and", "or", and "any other combination of all or some of the elements connected by said term".

[0168] The terms "less than" or "greater than" do not include specific numbers.

[0169] For example, less than 20 means less than the indicated number. Similarly, "more than" or "greater than" means more than or greater than the indicated number. For example, greater than 80% means greater than or more than the specified number 80%.

[0170] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean including the stated integer or step, or a collection of integers or steps, but not excluding any other integer or step, or a collection of integers or steps. When used herein, the term "comprising" can be replaced by the term "containing" or "including", or sometimes by the term "having" when used herein. "Consisting of" as used herein excludes any unstated element, step or ingredient.

[0171] The term "including" means "including but not limited to". "Including" and "including but not limited to" can be used interchangeably.

[0172] The term "about" means plus or minus 10%, preferably plus or minus 5%, more preferably plus or minus 2%, and most preferably plus or minus 1%.

[0173] In the description and claims of this specification, unless the context otherwise requires, the singular encompasses the plural. In particular, in the case of using an indefinite article, unless the context otherwise requires, the specification should be understood to contemplate both the plural and the singular.

[0174] It should be understood that the present invention is not limited to the specific methodologies, protocols, materials, reagents, substances, etc. described herein, as these can vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which is defined only by the claims.

[0175] All publications cited throughout this specification (including all patents, patent applications, scientific publications, specifications, etc.), whether before or after, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. In case of conflict or inconsistency between the material incorporated by reference and this specification, this specification will supersede any such material.

[0176] The contents of all documents and patent documents cited herein are hereby incorporated by reference in their entirety.

[0177] The present invention and its advantages can be better understood from the following examples provided for illustrative purposes only. These examples are not intended to limit the scope of the present invention in any way.

[0178] Embodiments of the present invention

[0179] The following examples illustrate the present invention but should not be construed as limiting the scope of the present invention.

[0180] Example 1

[0181] Materials and Methods

[0182] For the initial screening experiment, first generate hROSA-CAG-rtTA hiPSC-lines (such as Figure 2 A Figure 4 A showing the transfection of three plasmids expressing CAS9-nickase, two hROSA26-specific guide RNAs (SEQ ID NO: 66 and SEQ ID NO: 67), and a donor plasmid with a CAG-rtTA expression cassette; antibiotic selection, clonal expansion, and characterization of individual clone hiPSC colonies), and then perform four AAVS1 targeting vectors (SEQ ID NO: 61 to SEQ ID NO: 64) (see also Figure 4Transient transfection of (B-E) was performed to allow rapid overexpression of PU.1 (SEQ ID NO:2) alone or in combination with any one of three other transcription factors, RUNX1 (SEQ ID NO:4), CEBPB (SEQ ID NO:3), or IRF8 (SEQ ID NO:5), in a bicistronic expression cassette ( Figure 4 B-E) (SEQ ID NO:61 to SEQ ID NO:64). For screening purposes, the target cells were not clonally expanded, so overexpression occurred only in a subset of the cells.

[0183] Surprisingly, preliminary screening experiments demonstrated rapid induction of myeloid and microglial lineage markers in all three cell lines expressing PU.1 (SEQ ID NO:2) and any of the other three candidate reprogramming factors, but not in wild-type control hiPSCs or in cells expressing PU.1 (SEQ ID NO:2) alone.

[0184] Description

[0185] To develop a prototype protocol and establish appropriate readout parameters, the inventors decided to focus on the combinatorial overexpression of PU.1 (SEQ ID NO:2) and CEBPB (SEQ ID NO:3). Thus, fully validated inducible PU.1+CEBPB hiPSCs targeting dual GSH were generated and clonally expanded.

[0186] Observation

[0187] The addition of doxycycline led to a rapid loss of expression of the pluripotency markers OCT4 (SEQ ID NO:78) and NANOG (SEQ ID NO:79) and induction of the two transgenes in all cells (see Figure 6 ).

[0188] Example 2

[0189] Materials and Methods

[0190] Briefly, hiPSCs were plated as single cells onto Matrigel in pluripotency maintenance medium. Two days later, the medium was changed to Dulbecco's modified eagle medium (DMEM) / F12 supplemented with dox for transgene induction and small molecules and growth factors mimicking the above sequence of embryonic events. Three days after induction, the adherent cells began to delaminate from the tissue culture plate and were found as floating single cells in the supernatant.

[0191] Description

[0192] Subsequently, the present inventors conducted screening experiments for a longer period of time, in which cells were induced for up to 20 days to optimize the medium composition. Multicolor flow cytometry demonstrated a significant robust and rapid induction of myeloid cell surface markers, which were selected as a screening panel (CD11b (SEQ ID NO:45), CD14 (SEQ ID NO:47), CD45 (SEQ ID NO:50), CD163 (SEQ ID NO:53), CX3CR1 (SEQ ID NO:54)) for the induction of primitive macrophages and / or microglia. The inventors also noted important culture condition-dependent differences: the induction occurred most rapidly and efficiently when the overexpression of transcription factors was combined with timed exposure to extracellular signals (cues) (small molecules, growth factors) mimicking embryonic development: (1) patterning of pluripotent ectoderm (hiPSC) towards (posterior primitive streak) extraembryonic mesoderm and angioblasts, (2) induction of primitive hematopoiesis and early macrophage precursors, (3) differentiation towards primitive yolk sac macrophages, (4) differentiation towards microglia (see Figure 5 ).

[0193] Observation

[0194] As demonstrated by flow cytometry, the cells rapidly began to express typical myeloid surface proteins including CD45 (SEQ ID NO:50) (also known as PTPRC), CD11b (SEQ ID NO:45) (also known as ITGAM), CD14 (SEQ ID NO:47), and CX3CR1 (SEQ ID NO:54) (see Figure 5 B-C). By day 10, according to Muffat et al., 2016, all cells had transitioned into the supernatant and were plated onto poly-L-lysine (PLL)-coated tissue culture dishes in the final chemically defined microglia differentiation and maintenance medium. Interestingly, when doxycycline was withdrawn after the tenth day of the induction protocol, the differentiation towards microglia occurred even more efficiently, thus clearly demonstrating the independence of the cell phenotype from persistent transgene expression.

[0195] As quantified by flow cytometry (see Figure 5 c) or confirmed by immunocytochemistry (see Figure 5D), after 6 - 10 days of transgene - free differentiation and maturation in adherent culture, almost all cells expressed a wide range of common myeloid and more microglia - specific proteins, including CD39 (SEQ ID NO:49), P2RY12 (SEQ ID NO:56), TREM2 (SEQ ID NO:55), and TMEM119 (SEQ ID NO:57). Next, co - culture experiments were performed, in which the inventors plated microglial precursors onto a pure population of cortical neurons derived from syngeneic hiPSCs generated according to previously published protocols by Zhang et al., 2013 and Pawlowski et al., 2017. Compared to singly - cultured cells, microglia had a more branched (i.e., less activated) morphology (see Figure 5 E). Real - time qPCR analysis of hiPSCs and microglia in single culture confirmed the down - regulation of pluripotency factors, MYB - independence (consistent with microglia derived from primitive yolk - sac macrophages), and high expression of core microglial transcription factors, canonical surface markers, and recently proposed unique microglia - characteristic genes (see Figure 5 F).

[0196] References:

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Claims

1. A method for generating microglia from human-derived induced pluripotent stem cells, the method comprising the following steps: a) Targeted insertion of a nucleotide sequence encoding a transcriptional regulatory protein into a first genomic safe harbor locus; b) Targeted insertion of the coding sequence of the transcription factor PU.1 as shown in SEQ ID NO: 1 into a second, different genomic safe harbor locus, wherein the coding sequence is operably linked to an inducible promoter regulated by the transcriptional regulatory protein; insertion of the coding sequence of the transcription factor CEBPB as shown in SEQ ID NO: 3, the transcription factor RUNX1 as shown in SEQ ID NO: 4, or the transcription factor IRF8 as shown in SEQ ID NO: 5; and c) Culturing the stem cells harvested from steps a) and b) by exposure to at least one growth factor or small molecule that recapitulates signaling during at least one stage of embryonic development or adult microglial proliferation, differentiation, or polarization of microglia.

2. The method according to claim 1, wherein the transcriptional regulatory protein is a reverse tetracycline transactivator, and its activity is controlled by doxycycline or tetracycline.

3. The method according to claim 1, wherein the inducible promoter contains a Tet response element.

4. The method according to any one of claims 1 to 3, wherein the first and the second genomic safe harbor loci are selected from the hROSA26 locus, the AAVS1 locus, the CLYBL gene, the CCR5 gene, the HPRT gene, or genes having locus IDs 325 on chromosome 8, 227 on chromosome 1, 229 on chromosome 2, 255 on chromosome 5, 259 on chromosome 14, 263 on chromosome X, 303 on chromosome 2, 231 on chromosome 4, 315 on chromosome 5, 307 on chromosome 16, 285 on chromosome 6, 233 on chromosome 6, 311 on chromosome 134, 301 on chromosome 7, 293 on chromosome 8, 319 on chromosome 11, 329 on chromosome 12, and 313 on chromosome X.

5. An engineered human-derived induced pluripotent stem cell comprising a heterologous coding sequence of the transcription factor PU.1 as shown in SEQ ID NO: 1 and a heterologous coding sequence of the transcription factor CEBPB as shown in SEQ ID NO: 3, the transcription factor RUNX1 as shown in SEQ ID NO: 4, or the transcription factor IRF8 as shown in SEQ ID NO:

5.

6. The engineered human-derived induced pluripotent stem cell according to claim 5, further comprising a heterologous coding sequence of a transcriptional regulatory protein.

7. The engineered human-derived induced pluripotent stem cells according to claim 6, wherein the coding sequence of the transcription factor PU.1 is operably linked to an inducible promoter, and the inducible promoter is regulated by a transcriptional regulatory protein.

8. The engineered human-derived induced pluripotent stem cells according to claim 6, wherein the transcriptional regulatory protein is a reverse tetracycline transactivator, and its activity is controlled by doxycycline or tetracycline.

9. The engineered human-derived induced pluripotent stem cells according to claim 7, wherein the inducible promoter comprises a Tet-responsive element.

10. The engineered human-derived induced pluripotent stem cells according to any one of claims 5 to 9, wherein the engineered human-derived induced pluripotent stem cells express at least one protein selected from ITGAM, ITGAX, CD14, CD16, ENTPD1, PTPRC, CD68, CSF1R, CD163, CX3CR1, TREM2, P2RY12, TMEM119, and HLA-DR.

11. Use of the engineered human-derived induced pluripotent stem cells according to any one of claims 5 to 10 in the preparation of a medicament for treating a disease, wherein the disease is selected from Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, autoimmune antibody-mediated encephalitis, stroke, and vasculitis.

12. Use of the engineered human-derived induced pluripotent stem cells according to any one of claims 5 to 10 for in vitro culture with brain organoids.

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