Rapid and deterministic generation of microglia from human pluripotent stem cells

Abstract

The present invention relates to a method for producing microglia from stem cells, the method comprising the steps of: a) targeted insertion of a nucleotide sequence encoding a transcription factor protein into a first safe harbor site on the genome; and b) targeted insertion of a coding sequence for the transcription factor PU.1 (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter regulated by the transcription factor protein; expression of PU.1 (SEQ ID NO: 2); and culturing the stem cells obtained from steps a) and b) while exposing them to at least one growth factor or small molecule, wherein the at least one growth factor or small molecule mimics signaling during at least one of the embryonic developmental stages of microglia or the proliferation, differentiation, or polarization stages of adult microglia. The present invention further relates to microglia obtained by the method of the present invention and various uses thereof. TIFF2022534260000011.tif45170

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for producing microglia from stem cells, comprising the steps of: targeted insertion of a nucleotide sequence encoding a transcription regulator protein into a first safe harbor site on the genome; targeted insertion of a coding sequence of the transcription factor PU.1 into a second safe harbor site on the genome, the gene being operably linked to an inducible promoter regulated by the transcription regulator protein; expression of PU.1; and culturing the stem cells obtained from steps a) and b) while exposing them to at least one growth factor or small molecule, the at least one growth factor or small molecule recapitulating signaling during at least one of the stages of embryonic development of microglia or the stages of proliferation, differentiation or polarization of adult microglia. The present invention further relates to microglia obtained by the method of the present invention, and various uses thereof. [Background technology]

[0002] 2. Background of the Invention Microglia are the resident immune cells of the central nervous system (CNS) [Schafer et al., 2015]. They originate from primitive yolk sac macrophages that arise during the first wave of primary hematopoiesis in early embryogenesis. Primitive yolk sac macrophages spread through the bloodstream and colonize the developing CNS as soon as the circulation is established. In contrast to tissue-resident macrophages in other organs, microglia are not replaced by fetal monocytes during later stages of embryogenesis [McGrath et al., 303; Ginhoux et al., 2010; Gomez Perdiguero et al., 2015]. After the establishment of the microglial population during early embryogenesis, microglia maintain themselves by local proliferation throughout their lifetime and are not replaced by cells derived from the bone marrow [Reu et al., 2017]. Microglia are uniformly distributed throughout the brain and spinal cord and play important roles in CNS development, maintenance, plasticity, and defense [Schafer et al., 2015]. In a healthy CNS, “quiescent” homeostatic microglia are highly ramified cells with small cell bodies and thin cellular processes. These processes of microglia are motile and continuously sample their environment to scan for internal or external danger signals (e.g., invading pathogens or signals produced locally by damaged or dying cells). Detection of such signals triggers microglial activation, including complete changes in microglial morphology, gene expression, and function. Upon activation, microglia retract their processes and adopt an ameboid-like appearance. They actively migrate toward CNS lesions, following chemotaxis gradients, and secrete inflammatory cytokines.

[0003] Through a broad repertoire of cell surface receptors, including receptors for neurotransmitters and cytokines, microglia communicate with neurons, other glial cells, and cells of the peripheral immune system [Kettenmann et al., 2011]. Given their diverse functions and their unique position as the representative immune system in the healthy CNS, it is not surprising that microglia are involved in the development and progression of many neurological diseases [Ransohoff et al., 2016].

[0004] More recently, single-cell transcriptome profiling 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 transcriptome signature in a small subset of microglia 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 occur as a response to altered brain homeostasis in neurodegeneration and follows a distinct transcriptional program that is temporally and spatially regulated [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 / exacerbating functions. The availability of healthy and diseased human microglia in vitro and in vivo may facilitate the identification of factors involved in both their beneficial and detrimental functions and may facilitate the development of strategies to restore the homeostatic microglial signature or induce the DAM microglial signature. This point may have led us to target microglia for the treatment of neurodegenerative diseases.

[0005] 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. Previously, low-efficiency isolation from neurosurgical specimens or from postmortem brain tissue was the only available access. Human pluripotent stem cells (hPSCs) are an unlimited and renewable source from which, in theory, all cell types of the human organism can be generated [Thomson et al., 1998]. The revolutionary discovery that human dermal fibroblasts can be readily converted into human induced pluripotent stem cells (hiPSCs), which exhibit the same properties as embryonic stem cells, allows the generation of autologous and custom-made cell types for regenerative medicine applications. For several important applications, including disease modeling, drug discovery, and cell transplantation, large-scale production of mature human cell types from hPSCs is required. Recently, the first hPSC differentiation protocol for the generation of microglia was published. This was based on the initial formation of embryoid bodies (EBs) cultured for several months in the same “neuroglial 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 promising publication provided an elaborate medium composition for the final maturation and maintenance of human microglia. However, the long duration of the protocol, the inadequate definition of the initial stages of differentiation (i.e., the EB-based intermediate stages hardly follow the embryonic principles), and the need for several mechanical manipulation steps for cell purification appear to hinder the wide application of this protocol.Subsequently, several other groups have demonstrated the generation of microglia-like cells from hPSCs by similar, but different, classical differentiation approaches [Abud et al., 2017; Takata et al., 2017; Haenseler et al., 2017; Pandya et al., 2017; Douvaras et al., 2017]. Nevertheless, the in vitro derivation of specific human cell types, including microglia, in the quantities and purity required for downstream applications remains a challenge, and alternative methods are currently being explored [Cohen et al., 2011]. In contrast to classical differentiation, a more recent manufacturing strategy is direct cell reprogramming [Ladewig et al., 2013]. This refers to the direct conversion of any cell type (typically dermal fibroblasts) into another cell type without progressing through a pluripotent intermediate. Although rapid routes for cell generation from readily available cell types have been provided, the yield and purity of the desired cell populations remain low and insufficient [Zhang et al., 2013]. Recently, a third route, called "forward programming," has been proposed to produce mature human cell types with unprecedented speed and efficiency [Zhang et al., 2013].

[0006] Forward programming as a method to directly convert pluripotent stem cells, including hPSCs, into mature cell types has been recognized as a powerful strategy to induce human cells. It involves the forced expression of key lineage transcription factors (or non-coding RNAs, including IncRNAs and microRNAs) to convert stem cells into specific mature cell types. Currently available forward programming protocols are mainly based on lentiviral transduction of cells, which leads to erratic expression or complete silencing of randomly inserted inducible cassettes. This creates a situation where additional purification steps are required to isolate subpopulations expressing the required transcription factors. Therefore, further improvements of these methods are clearly needed.

[0007] Any improvement of the described method must ensure that the stable transcription of the genetic material contained within the inducible cassette, such as a transgene, is resistant to silencing and other negative effects associated with the integration site. Silencing can be caused by multiple epigenetic mechanisms, including DNA methylation or histone modifications. With prior art methods based on lentiviral transduction, the resulting cells are a heterogeneous population with fully expressed, partially expressed or silenced transgenes. This is clearly not desirable for many applications. Viral vectors have a proven tendency to integrate their genetic material into transcriptionally active areas of the genome, thus increasing potential oncogenic events due to mutagenesis at the insertion. For many applications, it is desirable to control the transcription of the inserted genetic material in the cell, so that the inducible cassette can be initiated as needed and transcribed at a specific level, including a high level. This cannot be achieved if the insertion of the inducible cassette is random in the genome.

[0008] The issue of microglia being involved in brain tissue, both in some serious diseases and in a way that makes their isolation from living tissue difficult, has been addressed in several publications. To overcome this issue, for example, human stem cells are used to generate microglia or microglia-like cells by defined culture conditions [Muffat et al., 2016] or by co-culturing with neurons derived from stem cells [Haenseler et al., 2017; Takata et al., 2017]. These methods rely solely on exposure to growth factors and cytokines to differentiate stem cells into microglia.

[0009] Moreover, the need for this special cell type is huge since microglia play a key role in virtually all diseases of the central nervous system, including neurodegenerative, neuroinflammatory or autoimmune diseases, autoantibody-mediated encephalitis or infectious diseases, neurovascular diseases, stroke, traumatic brain injury, and cancer, but the exact mechanisms underlying their role in various diseases remain unclear. Prior art is in agreement that stem cell-derived microglia indeed recapitulate the disease phenotype of the original patient [Muffat et al., 2016; Abud et al., 2017; Takata et al., 2017]. Given this finding, the huge scientific gap on the involvement of microglia in several diseases could be overcome by generating microglia from stem cells. However, classical protocols for differentiation of 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 stable introduction of an inducible cassette into the genome of stem cells and by making the transcription of the inducible cassette and the transcription factors inserted thereby controllable. The potential of these transcription factors to function as reprogramming factors for generating microglia was not known before and is the inventors' unique finding. This allows people to generate pure microglia populations that express all of the surface markers and RNAs observed in natural microglia populations. Furthermore, this method can be used to differentiate microglia from human iPS cells of patients with neurodegenerative diseases, and thus allows analyzing cell populations that otherwise would have remained completely unavailable for medical testing. Thus, there is a strong need to produce mature human microglia from a readily available source. Therefore, the technical problem underlying the present application is to meet these needs. The technical problem is solved by providing the embodiments reflected in the claims, described in the description, and illustrated in the examples and figures provided below. Summary of the Invention

[0011] The present inventors 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) targeted insertion of a nucleotide sequence encoding a transcription regulator protein into a first safe harbor site on the genome; and b) targeted insertion of a coding sequence for transcription factor PU.1 (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter regulated by the transcription regulator protein; expression of PU.1 (SEQ ID NO: 2); and c) culturing the stem cells obtained from steps a) and b) while exposing them to at least one growth factor or small molecule, wherein the at least one growth factor or small molecule recapitulates signaling during at least one of the stages of embryonic development of microglia or the stages of proliferation, differentiation, or polarization of adult microglia.

[0013] In one embodiment of the method of the present invention, at least one of the growth factors or small molecules is selected from the group consisting of 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 a further embodiment of the method of the present invention, at least one of the growth factors is CSF1 (SEQ ID NO: 14) or IL-34 (SEQ ID NO: 15).

[0015] In additional embodiments of the methods of the present invention, at least one of the small molecules is CHIR99021, LY294002, or IDE1.

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

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

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

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

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

[0021] In an additional embodiment of the methods of the present invention, the transcription regulator protein is reverse tetracycline transactivator (rtTA) (SEQ ID NO: 20) and its activity is regulated by doxycycline or tetracycline.

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

[0023] In a further embodiment of the method of the present invention, the first and second safe harbor sites on the genome are selected from the group consisting of 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 a gene having site ID 325 on chromosome 8 (SEQ ID NO: 27), a gene having site ID 227 on chromosome 1 (SEQ ID NO: 28), a gene having site ID 229 on chromosome 2 (SEQ ID NO: 29), a gene having site ID 255 on chromosome 5 (SEQ ID NO: 30), a gene having site ID 259 on chromosome 14 (SEQ ID NO: 31), a gene having site ID 263 on chromosome X (SEQ ID NO: gene with site ID 303 on chromosome 2 (SEQ ID NO: 33), gene with site ID 231 on chromosome 4 (SEQ ID NO: 34), gene with site ID 315 on chromosome 5 (SEQ ID NO: 35), gene with site ID 307 on chromosome 16 (SEQ ID NO: 36), gene with site ID 285 on chromosome 6 (SEQ ID NO: 37), gene with site ID 233 on chromosome 6 (SEQ ID NO: 38), gene with site ID 311 on chromosome 134 (SEQ ID NO: 39), gene with site ID 301 on chromosome 7 (SEQ ID NO: 40), gene with site ID 293 on chromosome 8 (SEQ ID NO: 41), gene with site ID 319 on chromosome 11 (SEQ ID NO: a gene having site ID 329 (SEQ ID NO: 43) on chromosome 12, and a gene having site ID 313 (SEQ ID NO: 44) on chromosome X.

[0024] In another embodiment of the method of the present invention, the stem cell is a pluripotent stem cell, an induced pluripotent stem cell (iPSC), a neural progenitor cell, a hematopoietic stem cell, or an embryonic stem cell (ESC).

[0025] In a further embodiment of the method of the present invention, the stem cells are human or mouse stem cells.

[0026] The present invention also relates to microglial cells obtainable by any of the methods according to the invention, wherein preferably the microglia express at least one of the microglial surface proteins selected from the group consisting of 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).

[0027] In a further aspect of the invention, the microglial cells are for use in therapy.

[0028] The present invention further relates to the use of such microglial cells according to the invention for the in vitro diagnosis of a disease, preferably selected from the group consisting of diseases of the central nervous system, 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 is directed to the use of such microglial cells according to the invention for in vitro culturing with cerebral organoids. [The present invention 1001] 1. A method for generating microglia from stem cells, comprising the steps of: (a) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein into a first safe harbor site in the genome; and (b) targeted insertion of the coding sequence for transcription factor PU.1 (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter that is regulated by the transcription factor protein; expression of PU.1 (SEQ ID NO: 2); and (c) culturing the stem cells obtained from steps (a) and (b) with exposure to at least one growth factor or small molecule, which recapitulates signaling during at least one of the stages of embryonic development of microglia, or the stages of proliferation, differentiation, or polarization of adult microglia. [The present invention 1002] The method of the present invention 1001, wherein at least one of the growth factors or small molecules is selected from the group consisting of 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. [The present invention 1003] The method of any one of claims 1001 to 1002, wherein at least one of the growth factors is CSF1 (SEQ ID NO: 14) or IL-34 (SEQ ID NO: 15). [The present invention 1004] Any of the aforementioned methods of the invention, wherein at least one of the small molecules is CHIR99021, LY294002, or IDE1. [The present invention 1005] Any of the aforementioned methods of the invention, wherein the first and second safe harbor sites on the genome are different. [The present invention 1006] Any of the aforementioned methods of the invention, further comprising the insertion of the coding sequence of the gene for the transcription factor CEBPB (SEQ ID NO: 3) and expressing it. [The present invention 1007] Any of the aforementioned methods of the invention, further comprising inserting and expressing the coding sequence of the gene for the transcription factor RUNX1 (SEQ ID NO: 4). [The present invention 1008] Any of the aforementioned methods of the invention, further comprising the insertion of the coding sequence of the gene for the transcription factor IRF8 (SEQ ID NO: 5) and expressing it. [The present invention 1009] Any of the aforementioned methods of the invention, further comprising the insertion of the coding sequence of the gene for the transcription factor SALL1 (SEQ ID NO: 6) and expressing it. [The present invention 1010] Any of the aforementioned methods of the present invention, wherein the transcriptional regulator protein is reverse tetracycline transactivator (rtTA) (SEQ ID NO: 20) and its activity is regulated by doxycycline or tetracycline. [The present invention 1011] Any of the aforementioned methods of the invention, wherein the inducible promoter comprises a Tet responsive element (TRE) (SEQ ID NO:21). [The present invention 1012] Any of the methods of the invention, wherein the first and second safe harbor sites on the genome are selected from the group consisting of 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 a gene having site ID 325 on chromosome 8 (SEQ ID NO:27), a gene having site ID 227 on chromosome 1 (SEQ ID NO:28), a gene having site ID 229 on chromosome 2 (SEQ ID NO:29), a gene having site ID 255 on chromosome 5 (SEQ ID NO:30), a gene having site ID 259 on chromosome 14 (SEQ ID NO:31), a gene having site ID 263 on chromosome X (SEQ ID NO: gene with site ID 303 on chromosome 2 (SEQ ID NO: 33), gene with site ID 231 on chromosome 4 (SEQ ID NO: 34), gene with site ID 315 on chromosome 5 (SEQ ID NO: 35), gene with site ID 307 on chromosome 16 (SEQ ID NO: 36), gene with site ID 285 on chromosome 6 (SEQ ID NO: 37), gene with site ID 233 on chromosome 6 (SEQ ID NO: 38), gene with site ID 311 on chromosome 134 (SEQ ID NO: 39), gene with site ID 301 on chromosome 7 (SEQ ID NO: 40), gene with site ID 293 on chromosome 8 (SEQ ID NO: 41), gene with site ID 319 on chromosome 11 (SEQ ID NO: 42), a gene with site ID 329 on chromosome 12 (SEQ ID NO: 43), and a gene with site ID 313 on chromosome X (SEQ ID NO: 44). [The present invention 1013] Any of the methods of the present invention, wherein the stem cells are pluripotent stem cells, induced pluripotent stem cells (iPSCs), neural progenitor cells, hematopoietic stem cells, or embryonic stem cells (ESCs). [The present invention 1014] Any of the aforementioned methods of the present invention, wherein the stem cells are human or mouse stem cells. [The present invention 1015] Microglia obtained by any one of the methods of the present inventions 1001 to 1014, preferably expressing at least one of the microglial surface proteins selected from the group consisting of: 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). [The present invention 1016] 1015. Microglia of the present invention for use in therapy. [The present invention 1017] Use of the microglia of the present invention 1015 or 1016 for the in vitro diagnosis of a disease. [The present invention 1018] The use of microglia of the present invention 1017, wherein the disease is selected from the group consisting of: diseases of the central nervous system, 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. [The present invention 1019] Use of the microglia of the present invention 1015 or 1016 for in vitro culturing with brain organoids. [Brief description of the drawings]

[0030] [Figure 1] Figure 1 provides an overview of the major pathways to produce cells, including: reprogramming of somatic cells (fibroblasts) into induced pluripotent stem cells (iPSCs) using four defined transcription factors Klf4, Oct4, c-Myc, and Sox2; direct reprogramming, which directly converts somatic cells into the desired target cell type using defined transcription factors; classical differentiation approaches, which represent a stepwise conversion of pluripotent stem cells into the desired target cell; and forward programming, which directly converts hPSCs into the target cell type. (Abbreviations: TF = transcription factor, ESC = embryonic stem cell, iPSC = induced pluripotent stem cell (ESCs and iPSCs are collectively referred to as pluripotent stem cells (PSCs)). [Diagram 2] Figure 2 shows the targeting strategy used in the present invention. The dox-inducible Tet-ON system targets the human ROSA26 locus (CAG-rtTA) and AAVS1 site (TRE-EGFP) in hPSCs. (Abbreviations: HAR = homology 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) [Diagram 3] FIG. 3 shows a table of key transcription factors of the microglial lineage selected as candidate reprogramming factors, the length of their coding sequences, and their sources. [Figure 4]Figure 4 shows the donor plasmids that were generated by molecular cloning and used to genetically modify either ROSA26 GSH or AAVS1 GSH. (Abbreviations: HAR = homology 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). [Figure 5A]Figure 5 shows an overview of the microglia forward programming protocol (see Figure 5A). Time course of cell surface markers expressed in primitive macrophages and microglia assessed by flow cytometry (n = 2 biological replicates) (see Figure 5B and Figure 5C). Microglia monoculture at day 20: Phase contrast live imaging of microglia-like cells and ICC for the microglia signature transmembrane protein TMEM119, for which a dedicated labeled flow antibody is not available (see Figure 5D). Microglia / neuron coculture at day 20: ICC for the intracellular calcium binding protein IBA1 (also known as AIF1) and the neuronal marker βIII-tubulin (TUBB3) (see Figure 5E). QPCR (Syber Green) of hiPSCs and microglia in monoculture (day 20). All values ​​are relative to the housekeeping gene GAPDH and normalized to hiPSCs. For the SPI1 and CEBPB transcripts, two different primer pairs (see SEQ ID NOs:80-87; SEQ ID NO:80: SPI1 full length forward primer; SEQ ID NO:81: SPI1 full length reverse primer; SEQ ID NO:82: SPI1 internal forward primer; SEQ ID NO:83: SPI1 internal reverse primer; SEQ ID NO:84: CEBPB full length forward primer; SEQ ID NO:85: CEBPB full length reverse primer; SEQ ID NO:86: CEBPB internal forward primer; SEQ ID NO:87: CEBPB internal reverse primer) were used to detect either the full length transcript (full length) or only the transcript from the respective internal locus and not the AAVS1 targeted transgene (internal). As expected, because transgene expression had ceased (by removal of dox on day 10 of the protocol), no differences were detected in relative expression levels, thus demonstrating that the cell phenotype was transgene-independent (F). [Figure 5B]See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 5E] See legend to Figure 5A. [Figure 5F] See legend to Figure 5A. [Figure 6] Figure 6 shows immunohistochemical analysis of a dual-targeted iPS cell line induced with doxycycline for 24 hours. Cells were positive for PU.1 and CEBPB but negative for OCT4. [Figure 7] FIG. 7 shows a map of the donor plasmid pUC_AAVS1_p-Resp-(PU.1-CEBPB) (SEQ ID NO: 61) for genetic modification of the AAVS1 locus, which contains the coding sequences for the transcription factors PU.1 and CEBPB. [Figure 8] FIG. 8 shows a map of the donor plasmid pUC_AAVS1_p-Resp-(PU.1-IRF8) (SEQ ID NO: 62) for genetic modification of the AAVS1 locus, which contains the coding sequences for the transcription factors PU.1 and IRF8. [Figure 9] FIG. 9 shows a map of the donor plasmid pUC_AAVS1_p-Resp-(PU.1-RUNX1) (SEQ ID NO: 63) for genetic modification of the AAVS1 locus, which contains the coding sequences for the transcription factors PU.1 and RUNX1. [Figure 10] FIG. 10 shows a map of the donor plasmid pUC_AAVS1_p-Resp-(PU.1) (SEQ ID NO: 64) for genetic modification of the AAVS1 locus, which contains the coding sequence for the transcription factor PU.1. [Figure 11] FIG. 11 shows a map of the donor plasmid pUC_AAVS1_p-Resp-(PU.1-SALL1) (SEQ ID NO: 65) for genetic modification of the AAVS1 locus, which contains the coding sequences for the transcription factors PU.1 and SALL1. [Figure 12] FIG. 12 shows a map of the plasmid ROSA-guideA_Cas9n (SEQ ID NO: 66), which contains the coding sequence for the Cas enzyme and guide RNA A. [Figure 13] FIG. 13 shows a map of the plasmid ROSA-guideB_Cas9n (SEQ ID NO: 67), which contains the coding sequence for the Cas enzyme and guide RNA B. [Figure 14] FIG. 14 shows a map of the donor plasmid pUC_ROSA_n_CAG-rtTA (SEQ ID NO: 72), which contains a constitutive CAG promoter and rtTA. [Figure 15] FIG. 15 shows a map of plasmid pZFN-AAVS1-L_ELD (SEQ ID NO: 68). [Figure 16] FIG. 16 shows a map of plasmid pZFN-AAVS1-R_KKR (SEQ ID NO: 69).

[0031] 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: homology arms (right, left), AmpR: ampicillin resistance gene, Ori: origin of replication, NeoR: neomycin resistance gene, KanR: kanamycin resistance gene. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Description of the Invention The present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeted insertion of a nucleotide sequence encoding a transcription regulator protein into a first safe harbor site on the genome; and b) targeted insertion of a coding sequence for transcription factor PU.1 (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter regulated by the transcription regulator protein; expression of PU.1 (SEQ ID NO: 2); and c) culturing the stem cells obtained from steps a) and b) while exposing them to at least one growth factor or small molecule, wherein the at least one growth factor or small molecule recapitulates signaling during at least one of the stages of embryonic development of microglia or the stages of proliferation, differentiation, or polarization of adult microglia.

[0033] In one embodiment, the present invention relates to a method for producing microglia from stem cells, the method comprising the steps of: a) targeted insertion of a nucleotide sequence encoding a transcription regulator protein into a first safe harbor site on the genome; and b) targeted insertion of a coding sequence for transcription factor PU.1 (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter regulated by the transcription regulator protein; expression of PU.1 (SEQ ID NO: 2); and c) culturing the stem cells obtained from steps a) and b) while exposing them to at least one growth factor or small molecule, wherein the at least one growth factor or small molecule recapitulates signaling during at least one of the stages of embryonic development of microglia or the stages of proliferation, differentiation, or polarization of adult microglia.

[0034] In one embodiment, the present invention relates to a method for producing microglia from stem cells, the method comprising the steps of: a) targeted insertion of a nucleotide sequence encoding a transcription regulator protein into a first safe harbor site on the genome; and b) targeted insertion of a coding sequence for transcription factor PU.1 (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter regulated by the transcription regulator protein; expression of PU.1 (SEQ ID NO: 2); and c) culturing the stem cells obtained from steps a) and b) while exposing them to at least one growth factor or small molecule, wherein the at least one growth factor or small molecule recapitulates signal transduction during at least one stage of embryonic development of microglia.

[0035] In one embodiment, the present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeted insertion of a nucleotide sequence encoding a transcription regulator protein into a first safe harbor site on the genome; and b) targeted insertion of a coding sequence of transcription factor PU.1 (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter regulated by the transcription regulator protein; expression of PU.1 (SEQ ID NO: 2); and c) culturing the stem cells obtained from steps a) and b) while exposing them to at least one growth factor or small molecule, wherein the at least one growth factor or small molecule recapitulates signal transduction during at least one stage of embryonic development of microglia.

[0036] In one embodiment, the present invention also relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeted insertion of a nucleotide sequence encoding a transcription regulator protein into a first safe harbor site on the genome; and b) targeted insertion of a coding sequence of transcription factor PU.1 (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter regulated by the transcription regulator protein; expression of PU.1 (SEQ ID NO: 2); and c) culturing the stem cells obtained from steps a) and b) while exposing them to at least one growth factor or small molecule, wherein the at least one growth factor or small molecule recapitulates signaling during at least one stage of differentiation of adult microglia.

[0037] In a further embodiment, the present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeted insertion of a nucleotide sequence encoding a transcription regulator protein into a first safe harbor site on the genome; and b) targeted insertion of a coding sequence of transcription factor PU.1 (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter regulated by the transcription regulator protein; expression of PU.1 (SEQ ID NO: 2); and c) culturing the stem cells obtained from steps a) and b) while exposing them to at least one growth factor or small molecule, wherein the at least one growth factor or small molecule recapitulates signaling during at least one stage of adult microglia polarization.

[0038] In another embodiment, the present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeted insertion of a nucleotide sequence encoding a transcription regulator protein into a first safe harbor site on the genome; and b) targeted insertion of a coding sequence of transcription factor PU.1 (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter regulated by the transcription regulator protein; expression of PU.1 (SEQ ID NO: 2); and c) culturing the stem cells obtained from steps a) and b) while exposing them to at least one growth factor or small molecule, wherein the at least one growth factor or small molecule recapitulates the embryonic development of microglia.

[0039] In a further embodiment, the present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeted insertion of a nucleotide sequence encoding a transcription regulator protein into a first safe harbor site on the genome; and b) targeted insertion of a coding sequence of transcription factor PU.1 (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter regulated by the transcription regulator protein; expression of PU.1 (SEQ ID NO: 2); and c) culturing the stem cells obtained from steps a) and b) while exposing them to at least one growth factor or small molecule, wherein the at least one growth factor or small molecule mimics signaling during at least one of the stages of embryonic development of microglia or the stages of proliferation, differentiation, or polarization of adult microglia.

[0040] In a further embodiment, the present invention relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeted insertion of a nucleotide sequence encoding a transcription regulator protein into a first safe harbor site on the genome; and b) targeted insertion of a coding sequence of transcription factor PU.1 (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter regulated by the transcription regulator protein; expression of PU.1 (SEQ ID NO: 2); and c) culturing the stem cells obtained from steps a) and b) while exposing them to at least one growth factor or small molecule, wherein the at least one growth factor or small molecule mimics signal transduction during at least one stage of embryonic development of microglia.

[0041] In one embodiment, the present invention also relates to a method for generating microglia from stem cells, the method comprising the steps of: a) targeted insertion of a nucleotide sequence encoding a transcription regulator protein into a first safe harbor site on the genome; and b) targeted insertion of a coding sequence of transcription factor PU.1 (SEQ ID NO: 1) into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter regulated by the transcription regulator protein; expression of PU.1 (SEQ ID NO: 2); and c) culturing the stem cells obtained from steps a) and b) while exposing them to at least one growth factor or small molecule, wherein the at least one growth factor or small molecule recapitulates the embryonic development of microglia in vitro.

[0042] As used within the scope of the present invention, the term "microglia" refers to a mature cell type that is a unique cell population in the central nervous system. As defined in Comparative Anatomy and Histology, "Microglia are the resident histiocytic-type cell and the key innate immune effector of the CNS. They are often described as either resting (ie, ramified) or activated, but these terms fail to convey the dynamic remodeling of their fine processes and constitutive immune surveillance activity. (…) Evidence suggests that early microglia are derived from yolk sac progenitors." (Hagan et al., 2012). Microglia as intended develop during early embryonic stages and reside in the brain throughout adult life.

[0043] As used within the scope of the present invention, the term "generation of microglia" refers to the generation of mature cells (microglia) from stem cells, the cells being obtained by any of the methods of the present invention described herein.

[0044] As used within the scope of the present invention, the term "stem cell" refers to a cell type that can divide to produce more cells or develop into a cell with a specific purpose. In the present invention, the stem cells used can be pluripotent stem cells. Pluripotent stem cells have the ability to differentiate into most cells 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 the blastocyst, an early stage pre-implantation embryo. Induced pluripotent stem cells (iPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem cell-like state by forcing the expression of genes and factors important for maintaining the characteristics that define embryonic stem cells. In 2006, it was shown that the introduction of four specific genes that code for transcription factors can convert adult cells into pluripotent stem cells (Takahashi et al., 2006), but subsequent studies have reduced / altered the number of genes required. Oct-3 / 4 and several members of the Sox gene family have been identified as potentially important transcription regulators involved in the induction process. Additional genes, including some members of Klf family, some members of Myc family, Nanog, and LIN28, can increase induction efficiency.Examples of genes that can be included in 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.

[0045] When the cell modified by inserting the inductive cassette is used in a human patient, it may be preferable that the cell is an iPSC derived from the individual. The use of such autologous cells may eliminate the need to match cells to the recipient. Alternatively, commercially available iPSCs may be used, which are known to those skilled in the art. Alternatively, the cell may be a tissue-specific stem cell, which may also be autologous or provided. Suitable cells include epiblast stem cells, artificial neural stem cells, and other tissue-specific stem cells.

[0046] In some embodiments of the method of the present invention, it may be preferable that the stem cells used are embryonic stem cells or stem cell lines. A number of embryonic stem cell lines are currently available, for example, 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.

[0047] As used within the scope of the present invention, the term "targeted insertion" refers to insertion into a safe harbor (GSH) site on the genome, preferably specifically into the sequence of GSH as described elsewhere. Any suitable technique for inserting a polynucleotide into a specific sequence can be used, and some have been described in the art. Suitable techniques include any method known to those skilled in the art that allows the vector to be recombined into the gap and introduced into the desired position. Thus, the first important step of target-site specific genome modification is the creation of a double-stranded DNA break (DSB) at the genomic locus to be modified. Different mechanisms of cell repair can be utilized to repair DSB and introduce desired sequences, and these are the more error-prone non-homologous end joining repair (NHEJ); and the donor DNA template-mediated homology-directed repair (HR), which can be used to insert an inducible cassette.

[0048] Several technologies exist that allow for the creation of customized site-specific DSBs in genomes. Many of these involve the use of customized endonucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or the clustered regularly interspaced short palindromic repeats / CRISPR-associated protein (CRISPR / Cas9) system (Gaj et al., 2013). Zinc finger nucleases are artificial enzymes that are created by fusing a zinc finger DNA binding domain with the nuclease domain of the restriction enzyme Fokl. The latter has a non-specific cleavage domain that must dimerize to cleave DNA. This means that two ZFN monomers are required to allow the Fokl domain to dimerize and cleave 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 5-7 bp apart to allow optimal dimerization of the Fokl domain. In this way, the enzyme can cleave DNA at a specific site, and target specificity is increased by ensuring that two DNA binding events in close proximity must occur to achieve a double-stranded break. Transcription Activator-Like Effector Nucleases, or TALENs, are dimeric transcription factors / nucleases. They are created by fusing a TAL effector DNA binding domain to a DNA cleavage domain (nuclease). Transcription Activator-Like Effectors (TALENs) can be designed to bind virtually any desired DNA sequence, and when combined with a nuclease, can cleave DNA at a specific location.TAL effectors are proteins secreted by Xanthomonas bacteria, whose DNA-binding domains contain repeats of a highly conserved sequence of 33-34 amino acids that differ at the 12th and 13th amino acids. The two positions are highly variable and show a strong correlation for the recognition of specific nucleotides. This clear association between amino acid sequence and DNA recognition makes it possible to design specific DNA-binding domains by selecting a combination of repeat segments that contain appropriate residues at the two variable positions. Thus, TALENs are composed of an array of 33-35 amino acid modules, each of which targets a single nucleotide. By selecting the array of modules, almost any sequence can be targeted. The nuclease used can be Fokl or a derivative thereof.

[0049] Three types of CRISPR machinery have been identified, of which type II is the most studied. The CRISPR / Cas9 system (type II system) utilizes the Cas9 nuclease to create double-stranded 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. CRISPRs are segments of prokaryotic DNA that contain short repeats of base sequences. Each repeat is followed by a short segment of "protospacer DNA" that originates from previous exposure to foreign genetic elements. The CRISPR spacer recognizes and cleaves foreign genetic elements using RNA interference. The CRISPR immune response occurs through two steps: CRISPR-RNA (crRNA) biogenesis and crRNA-guided interference. CrRNA molecules consist of a variable sequence transcribed from the protospacer DNA and CRISP repeats. Each crRNA molecule then hybridizes with a second RNA known as a trans-activating CRISPR RNA (tracrRNA), and finally, these two together form a complex with the nuclease Cas9. Sections of the crRNA encoded in the protospacer DNA cause Cas9 to cleave complementary target DNA sequences if they are adjacent to a short sequence known as a protospacer adjacent motif (PAM). This natural system has been engineered and exploited to introduce DSB breaks at specific sites in genomic DNA in many other applications. In particular, the CRISPR type II system from Streptococcus pyogenes can be used. Briefly, the CRISPR / Cas9 system includes two components that are delivered to cells to effect genome editing: the Cas9 nuclease itself, and a small guide RNA (gRNA). The gRNA is a fusion of a customized site-specific crRNA (directed to the target sequence), and a standardized tracrRNA.

[0050] Once DSB is created, a donor template with homology to the target locus is provided. DSB can be repaired by the homology-directed repair (HDR) pathway, which allows accurate insertion. Derivatives from this system are also possible. Mutant forms of Cas9 are available, such as Cas9D10A, which only has nickase activity. This means that the mutant form only cuts one DNA strand and does not activate NHEJ. Alternatively, when a homology repair template is provided, DNA repair is carried out only through the high-precision HDR pathway. Cas9D10A can be used as a pair of Cas9 complexes designed to nick adjacent DNA, with two types of sgRNAs complementary to the adjacent regions on opposite strands of the target site, which can be particularly beneficial. The elements for creating double-stranded DNA breaks can be introduced into one or more vectors, such as plasmids, for expression in cells. Therefore, any method for making specific targeted double-stranded break in genome to allow insertion of nucleotide sequence / gene / inducible cassette can be used in the method of the present invention.The method of the present invention may be preferred to use any one or more of ZFN, TALEN and / or CRISPR / Cas9 system or any derivative thereof to insert gene / inducible cassette.

[0051] Once DSB is created by any suitable means, the gene / inducible cassette for insertion can be provided in any suitable manner, as described below.The gene / inducible cassette and related genetic material form the donor DNA for repairing DNA at DSB, and it is inserted using standard cell repair machinery / pathway.As mentioned above, which path to use to repair the damage depends on how the cut is initiated.However, this is also within the knowledge of those skilled in the art.

[0052] As used within the scope of the present invention, the term "gene" refers to the basic physical unit of heredity, the linear sequence of nucleotides along a segment of DNA that provides coded instructions for synthesizing RNA that, when translated into protein, results in the expression of a hereditary trait.

[0053] As used within the scope of the present invention, the term "nucleotide sequence" refers to the succession of bases in a DNA segment forming a gene as defined above.

[0054] As used within the scope of the present invention, the term "transcriptional regulator protein" refers to a protein that binds to DNA, preferably in a sequence-specific manner, to a DNA site located in or near a promoter, and that promotes the binding of the transcriptional apparatus to the promoter and thus either promotes the transcription of the DNA sequence (transcriptional activator) or blocks this process (transcriptional repressor). Such entities are also known as transcription factors. The DNA sequences to which transcriptional regulator proteins bind are called transcription factor binding sites or response elements, and these are found in or near the promoter of the DNA sequence to be regulated. Responsive elements are part of the present invention. Transcriptional activator proteins bind to response elements and promote gene expression. Such proteins are preferred for controlling the expression of an inducible cassette in the method of the present invention. Transcriptional repressor proteins bind to response elements and prevent gene expression. Transcriptional regulator proteins can be activated or inactivated by several mechanisms, including binding of substances, interaction with other transcription factors (e.g. homo- or heterodimerization) or with co-regulatory proteins, phosphorylation, and / or methylation. Transcriptional regulators can be controlled by activation or inactivation. When transcriptional regulator proteins are transcriptional activator proteins, it is preferred that the transcriptional activator proteins require activation. This activation can be via any suitable means, but it is preferred that the transcriptional regulator proteins are activated by adding an exogenous substance to stem cells. The supply of exogenous substances to stem cells can be controlled, and thus the activation of the transcriptional regulator proteins can be controlled. Such transcriptional regulator proteins are also called inducible transcriptional regulator proteins.

[0055] As used within the scope of the present invention, the term "transcription factor" refers to a protein that binds to DNA, preferably a protein that binds in a sequence-specific manner to a DNA site located in or near a promoter, and that promotes the binding of the transcription apparatus to the promoter and thus either promotes the transcription of the DNA sequence (transcription activator) or blocks this process (transcription repressor). In the context of the present invention, a transcription factor is a desired genetic sequence, preferably a DNA sequence that is transferred into a cell together with an inducible cassette. The introduction of an inducible cassette into the genome has the potential to change the phenotype of the cell by adding a genetic sequence that allows gene expression. The method of the present invention provides for the controllable transcription in a cell of the genetic sequence of a set of transcription factors in an inducible cassette.

[0056] A master regulator is one or more of the following: a transcription factor, a transcription regulator, a cytokine receptor, or a signaling molecule, etc. A 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 the lineage of a cell. As used herein, a master regulator gene expressed at the initiation of a developing lineage or cell type participates in the specification of the lineage by regulating multiple downstream genes, either directly or through a cascade of changes in gene expression. When a master regulator is expressed, it has the ability to respecify the fate of cells that are 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 is 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).

[0057] As used within the scope of 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 integrating oncogene Spi1, Spleen Focus Forming Virus (SFFV) proviral integrating oncogene, or 31 kDa-Transforming Protein, SFPI1, SPI-1, SPI-A, PU.1, or OF, where "SPI1" refers to its gene (SEQ ID NO: 1) (Spi-1 proto-oncogene), which encodes an ETS domain transcription factor that activates gene expression during myeloid and B lymphoid cell development.

[0058] As used within the scope of the present invention, the term "genomic safe harbor site" refers to a genetic site where genetic material can be inserted without harmful effects on cells and where the inserted genetic material can be transcribed. Those skilled in the art may use these simplified and / or more formal criteria to identify suitable GSHs. Specific insertion into a genomic safe harbor site (GSH) is preferred over random integration into the genome, because it is expected to be a safer genome modification and is less likely to cause unwanted side effects, such as silencing natural gene expression or causing mutations that lead to cancerous cell types. Thus, a genomic safe harbor site is a locus in the genome where a gene or other genetic material can be inserted without harmful effects on cells or the inserted genetic material. Most beneficial are GSH sites where the expression of the inserted gene sequence is not disrupted by either read-through expression from adjacent genes or expression of an inducible cassette, and where there is minimal interference with the endogenous transcription program. More formal criteria have been proposed to help determine whether a particular locus is a GSH site (Pellenz et al., 2019). These criteria include the site being (i) > 300 kb from any cancer-related gene on the pan-cancer gene list, (ii) > 300 kb from any miRNA / other functional small RNA, (iii) > 50 kb from the 5' end of any gene, (iv) > 50 kb away from any replication origin, (v) > 50 kb away from any ultraconserved element, (vi) low transcriptional activity (no mRNA within ± 25 kb), (vii) not within a copy number variation region, (viii) within open chromatin (DHS signal within ± 1 kb), and (ix) unique (one copy in the human genome). Not all of these proposed criteria need to be met, as no GSH has been identified that meets all of these criteria.Preferably, a suitable GSH will meet at least 3, 4, 5, 6, 7, or 8 of these criteria, and most preferably will meet 9 of these criteria.

[0059] In the method of the present invention, the insertion occurs in different GSH. At least two GSH are required. The first GSH is modified by inserting a transcription regulator protein. The second GSH is modified by inserting an inducible cassette, where the cassette comprises a coding sequence operably linked to an inducible promoter. Other genetic material can also be inserted into either or both of these elements. The genetic sequence operably linked to the inducible promoter in the inducible cassette is preferably a DNA sequence. The genetic sequence of the inducible cassette preferably codes for an RNA molecule and is therefore capable of being transcribed. The transcription is controlled using an inducible promoter. The RNA molecule can be any sequence, but is preferably an mRNA that codes for a protein, shRNA, or gRNA.

[0060] The first GSH can be any suitable GSH site. Optionally, it is GSH with a constitutively expressed endogenous promoter, which causes the inserted transcription regulator to be constitutively expressed. Suitable GSH is hROSA26 site for human cells. In a further embodiment of the present invention, the inserted transcription regulator protein that is functionally linked to the promoter is functionally linked to a constitutive promoter. A constitutive promoter can be used, for example, when inserting at the hROSA26 site.

[0061] As used within the scope of the present invention, the term "inducible promoter" refers to a nucleotide sequence that initiates and regulates the transcription of a polynucleotide. An "inducible promoter" is a nucleotide sequence in which the expression of a genetic sequence operably linked to the promoter is controlled by an analyte, a cofactor, a regulatory protein, etc. In one embodiment of the method of the present invention, the control is influenced by a transcription regulator protein. The term "promoter" or "control element" is intended to include the full length promoter region and functional (e.g., transcription or translation control) segments of these regions. The gene encoding the transcription regulator protein is preferably operably linked to a constitutive promoter. Alternatively, the first GSH may be selected such that it already has a constitutive promoter that is also capable of driving the expression of the gene of the transcription regulator protein and any associated genetic material. A constitutive promoter ensures a sustained and high level of gene expression. Commonly used constitutive promoters include the human β-actin promoter (ACTB), the cytomegalovirus (CMV) promoter, the elongation factor-la (EFla) promoter, the phosphoglycerate kinase (PGK) promoter, and the ubiquitin C (UbC) promoter. The CAG promoter is a strong synthetic promoter that is often used to drive high levels of gene expression.

[0062] As used within the scope of the present invention, the term "culture" refers to the propagation of microorganisms, such as bacteria and yeasts, or human, plant or animal cells, under suitable conditions that ensure growth, known to those skilled in the art.

[0063] As used within the scope of the present invention, the term "growth factor" refers to a signaling molecule that controls the activity of cells in an autocrine, paracrine, or endocrine manner. As used herein, the term "growth factor" can be used interchangeably with "cytokine" in the context of the present invention. Growth factors or cytokines are produced by various cell types of organisms and exert their biological functions by binding to specific receptors and activating associated downstream signaling pathways, and they regulate gene transcription in the nucleus and ultimately stimulate biological responses including regulatory cellular processes such as cell division, cell survival, cell differentiation, adhesion, and migration.

[0064] As used within the scope of the present invention, the term "small molecule" refers to a biologically active molecule that is naturally or artificially produced, and can diffuse through cell membranes and modulate signal transduction pathways.Small molecules preferably used within the scope of the present invention can inhibit phosphatidylinositol 3 kinase (PI3K) and glycogen synthase kinase 3, such as LY294002 and CHIR99021, respectively.

[0065] As used within the scope of the present invention, the term "recapitulates signal transduction" means to mimic, mimic or resemble the function of secreted molecules, such as growth factors and / or chemokines, that affect cells in their natural environment, and thereby make it possible to manipulate microglia by their action.

[0066] As used within the scope of the present invention, the term "mimic signal transduction" means to mimic, mimic, resemble or reproduce the function of secreted molecules, such as growth factors and / or chemokines, that affect cells in their natural environment and thereby make it possible to generate microglia by their action.

[0067] As used within the scope of the present invention, the term "embryonic development of microglia" refers to the stepwise transition of pluripotent stem cells into mature microglial cells following differentiation outcomes during microglial development during human embryonic, fetal and postnatal development, starting from the preimplantation blastocyst stage embryo to a fully established and self-maintaining microglial population.

[0068] As used within the scope of the present invention, the term "adult microglial proliferation" refers to any cell division process that results in mature microglial cells.

[0069] As used within the scope of the present invention, the term "adult microglial differentiation" refers to the differentiation of cells in a microglial progenitor state into an adult microglial cell type that encompasses the typical characteristics of homeostatic / quiescent microglial cells.

[0070] As used within the scope of the present invention, the term "adult microglial polarization" refers to the response of mature microglial cells to extracellular stimuli resulting from the extracellular environment, signals from damaged neurons or glial cells, respectively, or exposure to plasma proteins due to blood-brain barrier dysfunction. This response of microglia includes migration of microglial cells to the site of injury and can have either neuroprotective or neurotoxic effects.

[0071] Further, in one embodiment of the method of the present invention, at least one of the growth factors or small molecules is selected from the group consisting of 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.

[0072] Activin A (SEQ ID NO: 7), as used herein, refers to activin beta-A chain, EDF, erythroid differentiation protein, FRP, FSH releasing protein, INHBA, inhibin beta-A chain, inhibin beta-1. The protein encoded by this gene is a member of the transforming growth factor beta (TGF-β) family of proteins produced by pluripotent stem cells, endoderm, and mesoderm.

[0073] BMP4 (SEQ ID NO: 8), as used herein, refers to bone morphogenetic protein 4, which is 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 beta superfamily.

[0074] FGF (SEQ ID NO: 9) as used herein refers to fibroblast growth factor. The protein encoded by this gene is a member of a family of cell signaling proteins, as described, for example, in Hui et al., 2018.

[0075] VEGF-A (SEQ ID NO: 10), as used herein, 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.

[0076] LY294002, as used herein, refers to a potent cell-permeable inhibitor of phosphatidylinositol 3-kinase (PI3K), which acts on the ATP-binding site of the enzyme (Vlahos et al., 1994). Its chemical structure is shown below. TIFF0007648543000001.tif32128

[0077] CHIR99021, as used herein, refers to an aminopyrimidine derivative that is a highly potent inhibitor of glycogen synthase kinase 3, which inhibits GSK3β (IC 50 = 6.7 nM) and inhibited GSK3α (IC 50 = 10 nM) and functions as a WNT activator. Its chemical structure is shown below. TIFF0007648543000002.tif37128

[0078] SCF (SEQ ID NO: 11), as used herein, refers to stem cell factor, which is 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 an important role in regulating embryonic and adult hematopoiesis.

[0079] IL-3 (SEQ ID NO: 12), as used herein, 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 cytokine that promotes growth.

[0080] IL-6 (SEQ ID NO: 13), as used herein, refers to interleukin 6, which is also known as B cell stimulatory factor 2, CTL differentiation factor, hybridoma growth factor, interferon beta-2, interleukin-6, IFN-beta-2, IFNB2, BSF-2, CDF, interferon beta 2, B cell differentiation factor, interferon beta 2, interleukin BSF-2, BSF2, HGF, or HSF. The protein encoded by this gene is a cytokine that functions in inflammation and B cell maturation.

[0081] CSF1 (SEQ ID NO: 14), as used herein, refers to colony-stimulating factor 1, which is 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.

[0082] IL-34 (SEQ ID NO: 15), as used herein, refers to interleukin 34, which is also known as C16 or f77. The protein encoded by this gene is a cytokine that promotes the differentiation and survival of monocytes and macrophages through the colony-stimulating factor 1 receptor.

[0083] CSF2 (SEQ ID NO: 16), as used herein, refers to colony-stimulating factor 2, which is also known as Sargramostim, 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.

[0084] CD200 (SEQ ID NO: 17), as used herein, refers to the CD200 gene, which is also known as the CD200 molecule, the CD200 antigen, the antigen identified by the monoclonal antibody MRC OX-2, the OX-2 membrane glycoprotein, MOX1, MOX2, OX-2, or MRC. The protein encoded by this gene is a type I membrane glycoprotein that contains two extracellular immunoglobulin domains, one transmembrane domain, and one cytoplasmic domain.

[0085] CX3CL1 (SEQ ID NO: 18), as used herein, refers to the CX3CL1 gene, which is also referred to as C-X3-C motif chemokine ligand 1, Small Inducible Cytokine Subfamily D (Cys-X3-Cys), Member 1 (fractalkine, neurotactin), chemokine (C-X3-C motif) ligand 1, CX3C membrane-anchored chemokine, Small Inducible Cytokine D1, C-X3-C motif chemokine 1, neurotactin, fractalkine, or SCYD1, NTT, Small Inducible Cytokine Subfamily D (Cys-X3-Cys), Member 1 (fractalkine, neurotactin), Also known as C3Xkine, ABCD-3, CXC3C, CXC3, NTN, or FKN. The protein encoded by this gene belongs to the CX3C subgroup of chemokines, which are characterized by the number of amino acids located between conserved cysteine ​​residues.

[0086] TGFβ1 (SEQ ID NO: 19), as used herein, refers to transforming growth factor β1, which is also known as transforming growth factor β-1 proprotein, preprotransforming growth factor β-1, TGFB, transforming growth factor β1, transforming growth factor β-1, latency-associated peptide, Kamurati-Engelman 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.

[0087] In a further embodiment of the method of the invention, at least one of the growth factors is CSF1 (SEQ ID NO: 14) or IL-34 (SEQ ID NO: 15). In a further embodiment of the method of the invention, at least one of the growth factors is CSF1 (SEQ ID NO: 14). In a further embodiment of the method of the invention, at least one of the growth factors is IL-34 (SEQ ID NO: 15).

[0088] In additional embodiments of the methods of the present invention, at least one of the small molecules is CHIR99021, LY294002, or IDE1.

[0089] LY294002, as used herein, refers to a potent cell-permeable inhibitor of phosphatidylinositol 3-kinase (PI3K), which acts on the ATP-binding site of the enzyme (Vlahos et al., 1994). Its chemical structure is shown below. TIFF0007648543000003.tif32128

[0090] CHIR99021, as used herein, refers to an aminopyrimidine derivative that is a potent inhibitor of glycogen synthase kinase 3, which inhibits GSK3β (IC 50 = 6.7 nM) and inhibited GSK3α (IC 50 = 10 nM) and functions as a WNT activator. Its chemical structure is shown below. TIFF0007648543000004.tif37128

[0091] IDE1, as used herein, refers to an inducer of definitive endoderm; a small molecule that activates the TGF-β pathway and can be used as an alternative to the growth factor TGF-β. Its chemical structure is shown below. TIFF0007648543000005.tif19128

[0092] In another embodiment of the method of the present invention, the first and second safe harbor sites on the genome are different.

[0093] In a further embodiment of the method of the invention, the method further comprises the insertion of the coding sequence of the gene for the transcription factor CEBPB (SEQ ID NO: 3) and its expression.

[0094] CEBPB (SEQ ID NO: 3), as used herein, refers to CCAAT enhancer binding protein beta, which is also known as CCAAT enhancer binding protein beta, CCAAT / enhancer binding protein (C / EBP) beta, interleukin 6-dependent DNA binding protein, CCAAT / enhancer binding protein beta, nuclear factor of interleukin 6, transcription factor 5, nuclear factor NF-IL6, TCF5, Liver-Enriched Transcriptional Activator Protein, CCAAT / enhancer binding protein beta, Liver-Enriched Inhibitory Protein, transcription factor C / EBP beta, Liver Activator Protein, C / EBP-beta, C / EBP beta, IL6DBP, NF-IL6, TCF-5, LAP, or LIP. This intronless gene encodes a transcription factor that contains a basic leucine zipper (bZIP) domain.

[0095] In another embodiment of the method of the present invention, the method further comprises the insertion of the coding sequence of the gene for the transcription factor RUNX1 (SEQ ID NO: 4) and its expression.

[0096] RUNX1 (SEQ ID NO:4), as used herein, means 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-alphaB, PEA2-alphaB, CBFA2, AML1, core binding factor Runt domain alpha subunit 2 core binding factor subunit alpha-2, AML1-EVI-1 fusion protein, Aml1 oncogene in acute myeloid leukemia, CBF-alpha-2, AML1-EVI-1, PEBP2alpha, CBF2alpha, PEBP2aB, AMLCR1, or EVI-1. The protein encoded by this gene is the alpha subunit of CBF, and it is believed to be involved in the progression of normal hematopoiesis.

[0097] In a further embodiment of the method of the invention, the method further comprises the insertion of the coding sequence of the gene for the transcription factor IRF8 (SEQ ID NO: 5) and its expression.

[0098] IRF8 (SEQ ID NO: 5), as used herein, refers to Interferon Regulatory Factor 8, which is 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), which is a transcription factor of the interferon (IFN) regulatory factor (IRF) family.

[0099] In another embodiment of the method of the invention, the method further comprises the insertion of the coding sequence of the gene for the transcription factor SALL1 (SEQ ID NO: 6) and its expression.

[0100] SALL1 (SEQ ID NO: 6), as used herein, refers to Spalt Like Transcription Factor 1, which is 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 Li 89, 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 may be part of the NuRD histone deacetylase complex (HDAC).

[0101] In an additional embodiment of the methods of the present invention, the transcription regulator protein is reverse tetracycline transactivator (rtTA) (SEQ ID NO: 20) and its activity is regulated by doxycycline or tetracycline.

[0102] As used within the scope of the present invention, the term "reverse tetracycline transactivator (rtTA)" refers to a transcription activator protein that is induced by tetracycline or its derivatives. Tetracycline-controlled transcription activation is a method of inducibly expressing genes, where transcription is reversibly started or stopped in the presence of the antibiotic tetracycline or one of its derivatives (e.g., the more stable doxycycline). In this system, the transcription activator protein can be a tetracycline-responsive transcription activator protein (rtTa), or a derivative thereof. The transcription regulator protein of the present invention can be rtTA. The rtTA protein can bind to DNA at a specific TetO operator sequence. Several repeats of such TetO sequences are placed upstream of a minimal promoter (such as the CMV promoter) to form together a tetracycline response element (TRE) (SEQ ID NO: 21). There are two formats of this system, depending on whether the addition of tetracycline or a derivative activates (Tet-On) or inactivates (Tet-Off) the rtTA protein. In the Tet-ON system, doxycycline activates the rtTA protein, which may also be used in one embodiment of the method of the present invention.

[0103] The Tet-On system is composed of two elements; (1) a constitutively expressed tetracycline-responsive transcription activator protein (rtTA), and a rtTA-sensitive inducible promoter (Tet-responsive element, TRE). To this, tetracycline or its more stable derivatives, including doxycycline (dox), can bind, causing activation of rtTA, allowing rtTA to bind to the TRE sequence and inducing expression of TRE-controlled genes. It is preferred to use this in the method of the present invention. Thus, the transcription regulator protein of the method of the present invention can be a tetracycline-responsive transcription activator protein (rtTA), which can be activated or inactivated by the exogenously supplied antibiotic tetracycline or one of its derivatives. When the transcription regulator protein is rtTA, the inducible promoter inserted into the second GSH site contains a tetracycline-responsive element (TRE). The exogenously supplied substance may be the antibiotic tetracycline or one of its derivatives such as doxycycline, preferably tetracycline or doxycycline.

[0104] Variants and modified rtTA proteins may be used in the methods of the invention, including Tet-On Advanced transactivator (also known as rtTA2S-M2) and Tet-On 3G (also known as rtTA-V16, derived from rtTA2S-S2).

[0105] In another embodiment of the methods of the present invention, the inducible promoter comprises a Tet responsive element (TRE) (SEQ ID NO: 21).

[0106] As used within the scope of the present invention, the term "Tet responsive element (TRE)" refers to the bacterial TetO sequence, which is a 7 repeat of 19 bp separated by a spacer sequence, also with a minimal promoter. The minimal promoter can be any suitable promoter, so that variants and modifications of the TRE sequence are available. Preferably, the minimal promoter shows no expression or a minimal expression level in the absence of rtTA binding. Thus, the inducible promoter inserted into the second GSH can include a TRE. The basic genetic principle underlying the present invention is also illustrated in Figure 2, which shows the different GSH sites (hROSA26 and AAVS1), as well as the rtTA (SEQ ID NO: 20) and TRE (SEQ ID NO: 21) that are incorporated.

[0107] In a further embodiment of the method of the present invention, the first and second safe harbor sites on the genome are selected from the group consisting of 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 a gene having site ID 325 on chromosome 8 (SEQ ID NO: 27), a gene having site ID 227 on chromosome 1 (SEQ ID NO: 28), a gene having site ID 229 on chromosome 2 (SEQ ID NO: 29), a gene having site ID 255 on chromosome 5 (SEQ ID NO: 30), a gene having site ID 259 on chromosome 14 (SEQ ID NO: 31), a gene having site ID 263 on chromosome X (SEQ ID NO: gene with site ID 303 on chromosome 2 (SEQ ID NO: 33), gene with site ID 231 on chromosome 4 (SEQ ID NO: 34), gene with site ID 315 on chromosome 5 (SEQ ID NO: 35), gene with site ID 307 on chromosome 16 (SEQ ID NO: 36), gene with site ID 285 on chromosome 6 (SEQ ID NO: 37), gene with site ID 233 on chromosome 6 (SEQ ID NO: 38), gene with site ID 311 on chromosome 134 (SEQ ID NO: 39), gene with site ID 301 on chromosome 7 (SEQ ID NO: 40), gene with site ID 293 on chromosome 8 (SEQ ID NO: 41), gene with site ID 319 on chromosome 11 (SEQ ID NO: a gene having site ID 329 (SEQ ID NO: 43) on chromosome 12, and a gene having site ID 313 (SEQ ID NO: 44) on chromosome X.Preferably, in a further embodiment of the method of the present invention, the first and second safe harbor sites on the genome are selected from the group consisting of: hROSA26 locus (SEQ ID NO: 22), AAVS1 locus (SEQ ID NO: 23), CLYBL gene (SEQ ID NO: 24), CCR5 gene (SEQ ID NO. 25), HPRT gene (SEQ ID NO. 26). More preferably, the first and second safe harbor sites on the genome are selected from the group consisting of hROSA26 locus (SEQ ID NO: 22) and AAVS1 locus (SEQ ID NO: 23).

[0108] Additional sites may be identified by searching for sites where the virus naturally integrates without interfering with native gene expression. In the context of the methods of the present invention, several GSH sites may be used, which are described in more detail below.

[0109] The adeno-associated virus integration site 1 locus (AAVS1) (SEQ ID NO: 23) is located in the protein phosphatase 1 regulatory subunit 12C (PPP1R12C) gene on human chromosome 19, which is uniformly and universally expressed in human tissues. This site acts as an integration site specific for AAV serotype 2, and has therefore been identified as a potential GSH. AAVS1 has been shown to be a favorable environment for transcription, as it contains an open chromatin structure and a natural chromosomal insulator that can make inducible cassettes resistant to silencing. There are no known adverse effects on cells due to the disruption of the PPP1R12C gene. Furthermore, inducible cassettes inserted into this site remain transcriptionally active in many diverse cell types. Therefore, AAVS1 is considered as a GSH and is widely used for targeted genetic modification in the human genome.

[0110] The hROSA26 locus (SEQ ID NO: 22) was identified based on sequence similarity to mouse GSH (ROSA26 - reverse oriented splice acceptor site #26). Although an orthologous site has been identified in humans, the site is not commonly used for the insertion of inducible cassettes. The inventors of the present invention have used a targeting system specific for the hROSA26 locus and have therefore been able to insert genetic material into this locus. The hROSA26 locus (SEQ ID NO: 22) is located on chromosome 3 (3p25.3) and can be found in the Ensembl database (GenBank: CR624523). The exact genomic coordinates of the integration site are 3:9396280-9396303: Ensembl. The integration site is located within the open reading frame (ORF) of the long non-coding RNA (reverse strand) of THUMPD3. Because the hROSA26 site has an endogenous promoter, the inserted genetic material may utilize the endogenous promoter or, alternatively, may be inserted operably linked to a promoter.

[0111] Intron 2 of the citrate lyase beta-like (CLYBL) gene (SEQ ID NO: 24) on the long arm of chromosome 13 has been identified as a suitable GSH because it is one of the identified integration hotspots of the phage-derived phiC31 integrase. Studies have demonstrated that an inducible cassette randomly inserted into this locus is stable and expressed. It has been shown that the insertion of an inducible cassette in this GSH does not disrupt neighboring gene expression (Cerbibi et al., 2015). Thus, CLYBL provides a GSH that can be used in the methods of the present invention.

[0112] CCR5 (SEQ ID NO: 25) is a gene that is located on chromosome 3 (position 3p21.31) and codes for the main co-receptor of HIV-1. Interest in using this site as GSH arises from null mutations in this gene that do not appear to have any adverse effects but render it resistant to HIV-1 infection. Zinc finger nucleases that target the third exon have been developed, which allow genetic material to be inserted into this locus. Given that the native function of CCR5 has not yet been elucidated, this site remains a putative GSH but can be used in the methods of the present invention. The hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene codes for a transferase enzyme that plays a central role in the production of purine nucleotides through the purine salvage pathway. It is discussed as a GSH site. Insertion at this site may be more suitable for mature cell types, such as for modification for gene therapy. GSH has been identified in other organisms and this includes the ROSA26, HRPT, and Hippll (Hll) loci in mice.

[0113] Mammalian genomes may contain GSH sites based on pseudo attP sites. For such sites, the Streptomyces phage-derived recombinase hiC31 integrase has been developed as a non-viral insertion tool due to its ability to integrate plasmids carrying attB sites containing inducible cassettes into pseudo attP sites. GSH is also present in plant genomes, and plant cell modification can be used in the methods of the present invention. GSH has been identified in the rice genome (Cantos et al., 2014).

[0114] The following SHS moieties may be used in any of the methods of the invention. They were published by Pellenz et al., 2019 and fulfill five of the nine criteria listed above: site ID 325 (SEQ ID NO: 27) on chromosome 8 at 68,720,172-68,720,191; site ID 227 (SEQ ID NO: 28) on chromosome 1 at 231,999,396-231,999,415; site ID 229 (SEQ ID NO: 29) on chromosome 2 at 45,708,354-45,708,373; site ID 255 (SEQ ID NO: 30) on chromosome 5 at 19,069,307-19,069,326; site ID 259 (SEQ ID NO: 31) on chromosome 14 at 92,099,558-92,099,577. 31); chromosome X: site ID 263 (SEQ ID NO: 32) at 12,590,812-12,590,831; chromosome 2: site ID 303 (SEQ ID NO: 33) at 77,263,930-77,263,949; chromosome 2: site ID 317 (SEQ ID NO: 60) at 77,263,930-77,263,949; chromosome 4: site ID 231 (SEQ ID NO: 34) at 58,976,613-58,976,632; chromosome 5: site ID 315 (SEQ ID NO: 35) at 7,577,728-7,577,747; chromosome 16: site ID 19,323,777-19,323,796 307 (SEQ ID NO: 36); site ID 285 (SEQ ID NO: 37) in chromosome 6: 89,574,320-89,574,339; site ID 233 (SEQ ID NO: 38) in chromosome 6: 114,713,905-114,713,924; site ID 311 (SEQ ID NO: 39) in chromosome 6: 134,385,946-134,385,965; site ID 301 (SEQ ID NO: 40) in chromosome 7: 113,327,685-113,327,704; site ID 293 (SEQ ID NO: 41) in chromosome 8: 40,727,927-40,727,946;Chromosome 11: site ID 319 (SEQ ID NO: 42) at 32,680,546-32,680,565; Chromosome 12: site ID 329 (SEQ ID NO: 43) at 126,152,581-126,152,600; Chromosome X: site ID 313 (SEQ ID NO: 44) at 16,059,732-16,059,751.

[0115] In another embodiment of the method of the present invention, the stem cell is a pluripotent stem cell, an induced pluripotent stem cell (iPSC), a neural progenitor cell, a hematopoietic stem cell, or an embryonic stem cell (ESC).

[0116] Within the scope of the present invention, the term "pluripotent stem cells" is used as defined above.

[0117] As used within the scope of the present invention, the term "neural progenitor cell" refers to a multipotent cell state between pluripotent stem cells and mature somatic cells, which is usually committed to become specialized cell types such as neurons, oligodendrocytes, and astrocytes.

[0118] Within the scope of the present invention, the term "induced pluripotent stem cells (iPSCs)" is used as defined above.

[0119] As used within the scope of the present invention, the term "hematopoietic stem cell" refers to a stem cell that produces blood cells. This particular type of multipotent stem cell can produce any type of blood cell, but has lost the ability to produce other cell types.

[0120] Within the scope of the present invention, the term "embryonic stem cells (ESCs)" is used as defined above.

[0121] In a further embodiment of the method of the present invention, the stem cells are human or mouse stem cells.

[0122] As used within the scope of the present invention, the term "human or mouse stem cells" refers to cells derived from humans or mice. However, the stem cells used in the method of the present invention can be any cells of humans or animals. It is preferably a mammalian cell, for example, a cell derived from rodents, such as mice and rats; a cell derived from marsupials, such as kangaroos and koalas; a cell derived from non-human primates, such as bonobos, chimpanzees, lemurs, gibbons, and apes; a cell derived from camelids, such as camels and llamas; a cell derived from livestock animals, such as horses, pigs, cows, buffaloes, bisons, goats, sheep, deer, reindeer, donkeys, bantengs, yaks, chickens, ducks, and turkeys; a cell derived from domesticated animals, such as cats, dogs, rabbits, and guinea pigs. The cell is preferably a human cell. In one aspect, the cell is preferably derived from livestock animals. The type of cell used in the methods of the present invention will depend on the use of the cells once insertion of the genetic material into the GSH site is complete.

[0123] The present invention also relates to microglial cells obtainable by any of the methods according to the invention, wherein preferably the microglia express at least one of the microglial surface proteins selected from the group consisting of 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).

[0124] Thus, microglia are further defined by their expression of 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). ID NO: 58). These proteins are defined as follows:

[0125] ITGAM (CD11B), as used herein, refers to the integrin subunit αM, which is the gene encoding the integrin αM chain. Integrins are heterodimeric integral membrane proteins composed of an α chain and a β chain. The protein sequence is shown in SEQ ID NO: 45.

[0126] ITGAX (CD11C) as used within the scope of the present invention means integrin subunit αX, and the gene encodes the protein of the integrin αX chain, the protein sequence of which is shown in SEQ ID NO: 46.

[0127] CD14, as used herein, refers to monocyte differentiation antigen CD14, and the protein encoded by this gene is a surface antigen selectively expressed in monocytes / macrophages. The protein sequence is shown in SEQ ID NO: 47.

[0128] CD16, as used herein, refers to FCGR3A, Fc fragment of IgG receptor IIIa, and this 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. Its protein sequence is shown in SEQ ID NO: 48.

[0129] ENTPD1 (CD39), as used herein, 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 is shown in SEQ ID NO: 49.

[0130] PTPRC (CD45), as used herein, refers to type C receptor protein tyrosine phosphatase, and the protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family. The protein sequence is shown in SEQ ID NO: 50.

[0131] CD68, as used herein, refers to the CD68 antigen, and the gene encodes a 110-kD transmembrane glycoprotein that is highly expressed by human monocytes and tissue macrophages, the protein sequence of which is shown in SEQ ID NO:51.

[0132] CSF1R (CD115), as used herein, refers to colony-stimulating factor 1 receptor, and the protein encoded by this gene is the receptor for colony-stimulating factor 1, a cytokine that controls the production, differentiation, and function of macrophages. The protein sequence is shown in SEQ ID NO: 52.

[0133] CD163, as used herein, 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 exclusively in monocytes and macrophages. Its protein sequence is shown in SEQ ID NO: 53.

[0134] CX3CR1, as used herein, means C-X3-C motif chemokine receptor 1, and the protein encoded by this gene is the receptor for fractalkine. The protein sequence is shown in SEQ ID NO: 54. Fractalkine is a transmembrane protein and a chemokine involved in leukocyte adhesion and migration.

[0135] TREM2, as used herein, refers to Triggering Receptor Expressed On Myeloid Cells 2, and this gene encodes a membrane protein that forms a receptor signaling complex with the TYRO protein tyrosine kinase binding protein. The protein sequence is shown in SEQ ID NO: 55.

[0136] P2RY12, as used herein, refers to the purinergic receptor P2Y12, and the product of this gene belongs to the family of G protein-coupled receptors. Its protein sequence is shown in SEQ ID NO: 56.

[0137] TMEM119, as used herein, refers to a gene encoding a protein, transmembrane protein 119. Its associated pathways include microglial activation during neuroinflammation. Its protein sequence is shown in SEQ ID NO: 57.

[0138] HLA-DR, as used herein, refers to DRα and DRβ of class II of the major histocompatibility complex, and both HLA-DRA and HLA-DRB1 are paralogs of the HLA class II α chain, the protein sequence of which is shown in SEQ ID NO:58.

[0139] In a further aspect, the present invention also comprises a microglial cell according to the present invention for use in therapy.

[0140] As used in the present invention, the term "therapy" refers to any form of treating a disease or an undesired health condition of an organism, animal, or human. It may also include gene therapy, which may be defined as the deliberate insertion of foreign DNA into the nucleus of a cell for therapeutic purposes. Such definitions include: delivery of one or more genes to a cell to provide a wild-type version of a defective gene; addition of a gene for an RNA molecule that interferes with the expression of a target gene (which may be defective); delivery of suicide genes (such as thymidine kinase (HSV-tk), an enzyme of the herpes simplex virus that converts the harmless prodrug ganciclovir (GCV) into a cytotoxic drug, and cytosine deaminase (CD)); DNA vaccines for immunization or cancer therapy (including adoptive cellular immunotherapy); and any of the introduction of genes into a cell for therapeutic purposes. Another source of. In addition, mature microglia can be used directly for transplantation into the human or animal body. Alternatively, microglia can be used as test material for research, including the effects of drugs on gene expression and drug interactions with specific genes. Research microglia can involve the use of inducible cassettes with genetic sequences of unknown function to study the controllable expression of said genetic sequences. In addition, this can allow the use of microglia to produce large amounts of desired substances, such as growth factors or cytokines.

[0141] Furthermore, the present invention also relates in one embodiment to the use of such microglial cells according to the present invention for in vitro diagnosis of diseases.Preferably, the diseases are selected from the group consisting of diseases of the central nervous system, 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.

[0142] Furthermore, the present invention is directed to the use of such microglial cells according to the invention for in vitro culturing with cerebral organoids.

[0143] As used within the scope of the present invention, the term "organoid" refers to an in vitro 3D organ model derived from cells (mainly stem cells) and which, in combination with the microglia produced by the present invention, represents a powerful tool for medical diagnostics to study the involvement of microglia in and interactions with other cells of the brain.

[0144] As used herein, the singular forms "a," "an," and "the" are understood to include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "reagents" includes one or more of such various reagents, and reference to "methods" includes reference to equivalent steps and methods known to those of skill in the art that may be modified or substituted for the methods described herein.

[0145] Unless otherwise specified, the term "at least" preceding a series of elements is understood to refer to every element in the series. The term "at least one", unless otherwise specifically defined, refers to one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is intended that such equivalents are encompassed by the present invention.

[0146] Wherever used in this specification, the term "and / or" includes the meaning of "and," "or," and "all or any other combination of the elements connected by that term."

[0147] The term "less than" or, conversely, the term "greater than" is exclusive of numerals.

[0148] For example, less than 20 means less than the specified number. Similarly, "more" or "exceeding" means more or greater than a specified number, for example, more than 80% means more or greater than 80% of a specified number.

[0149] Throughout this specification and the claims appended hereto, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" are understood to mean the inclusion of a specified integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including," or, as used herein, can sometimes be replaced with the term "having." As used herein, "consisting of" does not include any elements, steps, or ingredients not specified.

[0150] The term "including" means "including, but not limited to." "Including" and "including, but not limited to" are used interchangeably.

[0151] The term "about" means plus or minus 10%, which is preferably plus or minus 5%, more preferably plus or minus 2%, and most preferably plus or minus 1%.

[0152] Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification is understood to contemplate plural as well as singular, unless the context requires otherwise.

[0153] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention, the scope of which is defined only by the claims.

[0154] All publications cited in the text of this specification, whether supra or infra, including all patents, patent applications, scientific publications, instructions, etc., are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that the material incorporated by reference is contradictory or inconsistent with the present specification, the present specification will take precedence over any such material.

[0155] The contents of all publications and patent documents cited herein are incorporated by reference in their entirety.

[0156] A better understanding of the present invention and its advantages will be gained from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any manner. EXAMPLES

[0157] Example of the invention The following examples illustrate the present invention, but the examples should not be construed as limiting the scope of the invention.

[0158] Example 1 material and method For the initial screening experiments, initial hROSA-CAG-rtTA hiPSC lines were generated (nucleofection of three plasmids expressing CAS9 nickase, two hROSA26-specific guide RNAs (SEQ ID NO: 66 and SEQ ID NO: 67), and a donor plasmid carrying the CAG-rtTA expression cassette as shown in Figure 2A and Figure 4A; antibiotic selection, clonal expansion, and characterization of clonal individual hiPSC colonies), followed by transient transfection of four vectors targeting AAVS1 (SEQ ID NO: 61 to SEQ ID NO: 64) (see also Figure 4B to 4E) to rapidly overexpress PU.1 (SEQ ID NO: 2) alone or in the form of a bicistronic expression cassette (Figure 4B to 4E) (SEQ ID NO: 61 to SEQ ID NO. 64). 2) in combination with one of three other transcription factors, RUNX1 (SEQ ID NO: 4), CEBPB (SEQ ID NO: 3), or IRF8 (SEQ ID NO: 5). For screening purposes, targeted cells were not clonal expanded, but rather overexpressed in a subset of cells.

[0159] Surprisingly, initial screening experiments demonstrated that myeloid and microglial lineage markers were rapidly induced in all three cell lines expressing PU.1 (SEQ ID NO: 2) plus 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.

[0160] explanation To develop a prototype protocol and establish appropriate readout parameters, we decided to focus on combinatorial overexpression of PU.1 (SEQ ID NO: 2) and CEBPB (SEQ ID NO: 3). Thus, fully validated, dual GSH-targeted, inducible PU.1 + CEBPB hiPSCs were generated and clonally expanded.

[0161] observation Addition of doxycycline resulted in rapid loss of expression of pluripotency markers OCT4 (SEQ ID NO: 78) and NANOG (SEQ ID NO: 79) and induction of both transgenes in all cells (see FIG. 6).

[0162] Example 2 material and method Briefly, hiPSCs were plated as single cells on Matrigel in pluripotency maintenance medium. After 2 days, the medium was replaced with Dulbecco's Modified Eagle Medium (DMEM) / F12, in which small molecules and growth factors were added to mimic the sequence of embryonic events outlined above, in addition to dox for transgene induction. After 3 days of induction, adherent cells began to detach from the tissue culture plate and were found as floating single cells in the supernatant.

[0163] explanation We then performed longer screening experiments in which cells were induced for up to 20 days to optimize the medium composition. Multicolor flow cytometry demonstrated a remarkably robust and rapid induction of myeloid cell surface markers (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)) that were selected as a screening panel for the induction of primitive macrophages and / or microglia. We also found important differences that were dependent on the culture conditions: induction occurred most rapidly and efficiently when overexpression of transcription factors was performed in conjunction with timed exposure to extracellular cues (small molecules, growth factors) that mimicked the following sequence of embryonic development: (1) patterning of pluripotent epiblasts (hiPSCs) toward extraembryonic mesoderm (of the posterior primitive streak) and hemangioblasts, (2) induction of primary hematopoietic and early macrophage progenitors, (3) differentiation into primitive yolk sac macrophages, and (4) differentiation into microglia (see FIG. 5).

[0164] observation As evidenced by flow cytometry, the cells rapidly started 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 Figures 5B-5C). All cells migrated into the supernatant by day 10 and were plated on poly-L-lysine (PLL)-coated tissue culture dishes in chemically defined definitive medium for microglial differentiation and maintenance according to Muffat et al., 2016. Interestingly, microglial differentiation occurred much more efficiently after doxycycline was removed on day 10 of the induction protocol, thus clearly demonstrating that the cell phenotype is independent of sustained transgene expression.

[0165] After 6-10 days of transgene-free differentiation and maturation in adherent culture, virtually all cells expressed a broad range of proteins common to myeloids and more specific to microglia, including CD39 (SEQ ID NO: 49), P2RY12 (SEQ ID NO: 56), TREM2 (SEQ ID NO: 55), and TMEM119 (SEQ ID NO: 57), as quantified by flow cytometry (see FIG. 5c) or evidenced by immunohistochemical analysis (see FIG. 5D). Co-culture experiments were then performed in which the inventors plated microglial progenitor cells onto pure populations of isogenic hiPSC-derived cortical neurons generated according to protocols previously published by Zhang et al., 2013 and Pawlowski et al., 2017. Microglial cells adopted a more branched (i.e., more inactive) morphology compared to cells in monoculture (see FIG. 5E). Real-time qPCR analysis of hiPSCs and microglia in monoculture demonstrated downregulation of pluripotency factors, MYB independence (consistent with microglia originating from primitive yolk sac macrophages), and high expression of microglial core transcription factors, classical surface markers, and recently suggested unique microglial signature genes (see FIG. 5F).

[0166] References: TIFF0007648543000006.tif217165TIFF0007648543000007.tif237165TIFF0007648543000008.tif237165TIFF0007648543000009.tif17165

Claims

1. A method for generating microglia from induced pluripotent stem cells, comprising the steps of: (a) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein into a first safe harbor site in a genome; and (b) targeted insertion of the coding sequence for the transcription factor PU.1 into a second safe harbor site on the genome, wherein the gene is operably linked to an inducible promoter that is regulated by the transcription factor protein; expression of PU.1; insertion of the coding sequence for the transcription factor CEBPB, the transcription factor RUNX1, or the transcription factor IRF8; and (c) culturing the induced pluripotent stem cells obtained from steps (a) and (b) with exposure to at least one growth factor or small molecule, which recapitulates signaling during at least one of the stages of embryonic development of microglia, or the stages of proliferation, differentiation, or polarization of adult microglia.

2. The method of claim 1, wherein at least one of the growth factors or small molecules is selected from the group consisting of activin A, BMP4, FGF, VEGF-A, LY294002, CHIR99021, SCF, IL-3, IL-6, CSF1, IL-34, CSF2, CD200, CX3CL1, TGFβ1, and IDE1.

3. The method of claim 1 or 2, wherein at least one of the growth factors is CSF1 or IL-34.

4. The method of any one of claims 1 to 3, wherein at least one of the small molecules is CHIR99021, LY294002, or IDE1.

5. The method of any one of claims 1 to 4, wherein the first and second safe harbor sites on the genome are different.

6. The method according to any one of claims 1 to 5, wherein the transcription regulator protein is reverse tetracycline transactivator (rtTA) and its activity is controlled by doxycycline or tetracycline.

7. The method of any one of claims 1 to 6, wherein the inducible promoter comprises a Tet responsive element (TRE).

8. The method of any one of claims 1 to 7, wherein the first and second safe harbor sites on the genome are selected from the group consisting of the hROSA26 locus, the AAVS1 locus, the CLYBL gene, the CCR5 gene, the HPRT gene, or the gene with site ID 325 on chromosome 8, the gene with site ID 227 on chromosome 1, the gene with site ID 229 on chromosome 2, the gene with site ID 255 on chromosome 5, the gene with site ID 259 on chromosome 14, the gene with site ID 263 on chromosome X, the gene with site ID 303 on chromosome 2, the gene with site ID 231 on chromosome 4, the gene with site ID 315 on chromosome 5, the gene with site ID 307 on chromosome 16, the gene with site ID 285 on chromosome 6, the gene with site ID 233 on chromosome 6, the gene with site ID 311 on chromosome 6, the gene with site ID 301 on chromosome 7, the gene with site ID 31 on chromosome 8, the gene with site ID 31 on chromosome 9, the gene with site ID 31 on chromosome 10, the gene with site ID 31 on chromosome 11, the gene with site ID 31 on chromosome 12, the gene with site ID 31 on chromosome 13, the gene with site ID 31 on chromosome 14, the gene with site ID 31 on chromosome 15, the gene with site ID 31 on chromosome 16, the gene with site ID 31 on chromosome 17, the gene with site ID 31 on chromosome 18, the gene with site ID 31 on chromosome 19, the gene with site ID 31 on chromosome 20, the gene with site ID 31 on chromosome A gene with site ID 293, a gene with site ID 319 on chromosome 11, a gene with site ID 329 on chromosome 12, and a gene with site ID 313 on the X chromosome.

9. A method according to any one of claims 1 to 8, wherein the induced pluripotent stem cells are human or mouse induced pluripotent stem cells.

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