Methods and compositions for producing microglial cells

Abstract

Provided herein are methods and compositions for differentiating induced pluripotent stem cells into microglial cell-like cells by overexpressing transcription factors (e.g., SPI1, CEBPA, FLI1, MEF2C, CEBPB, and / or IRF8).

Description

[0001] [Cross-reference to related applications]

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 333,573, filed on April 22, 2022, Vol. 35, No. 119(e), which is hereby incorporated by reference in its entirety.

[0003]

Electronic Sequence Listing Reference

[0004] The electronic sequence listing (contents of H049870762WO00-SEQ-KVC.xml; size: 13,069 bytes; and creation date: April 19, 2023) is incorporated herein by reference in its entirety.

[0005]

Government licensing rights

[0006] This invention was made with government support under Award HG008525 from the National Institutes of Health (NIH). The government has certain rights in this invention. [Background of the invention]

[0008] Microglia are the resident immune cells of the brain that originate from erythromyeloid progenitors (EMPs) in the yolk sac. They play important and diverse roles in brain development and maintaining homeostasis. Recent studies have shown a link between neuroinflammation and neurodegenerative diseases such as Alzheimer's disease (AD), and along these lines, microglia have been shown to be an important cell type in AD and other neurodegenerative diseases. Functional studies that define therapies targeting human microglia have been greatly hindered by the limited availability of human brain biopsies. The supply issue cannot be simply alleviated by using mouse models, as the differences between human and mouse microglia limit the transferability of knowledge. Generating human microglia-like cells from hiPSCs may fill this gap. Several studies have achieved this goal through a process of embryoid body formation, growth factor treatment, and in some cases co-culture with neurons. These protocols draw inspiration from the natural developmental stages of microglia, with timelines ranging from 30 to 74 days. [Summary of the invention]

[0010] Since the effects of exogenous factors on cell fate are often mediated by TFs, direct manipulation of TF expression can differentiate hiPSCs into microglia in a shorter time. The ability to differentiate stem cells into human cell types is an important tool for defining basic mechanisms and therapeutic approaches, especially for cell types that are usually not accessible by biopsy. However, although it has been found that the processed expression of transcription factors (TFs) identified by TF screening can quickly and efficiently generate certain cell types, the generation of types that require complex combinations of TFs has been elusive. Here, an iterative hybrid single-cell TF screening method was developed that uses the generation of human microglia-like cells as a test platform to improve the identification of effective TF combinations. For example, two iterations determined that the combination of SPI1, CEBPA, FLI1, MEF2C, CEBPB, and IRF8 was sufficient to differentiate human iPSCs into microglia-like cells in about 4 days. Characterization of TF-induced microglia showed molecular and functional similarities to primary microglia. The use of single-cell atlas reference datasets was explored to confirm the identified TFs and how combining single-cell TF perturbations and gene expression data can construct causal gene regulatory networks. This article describes what is needed to shape these approaches into a common integrated process, further enhancement ideas, and possible applications.

[0011] In some aspects, a pluripotent stem cell (PSC) is provided, comprising: one or more processed polynucleotides, wherein the processed polynucleotides comprise open reading frames encoding a SPI1 protein, a FLI1 protein, and a CEBPA protein.

[0012] In some embodiments, the one or more processed polynucleotides comprise a first polycistronic polynucleotide comprising: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the FLI1 protein, and a third open reading frame encoding the CEBPA protein.

[0013] In some embodiments, the one or more processed polynucleotides comprise a first polycistronic polynucleotide, which comprises the following open reading frames, ordered in the 5' to 3' direction of the first polycistronic polynucleotide: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the FLI1 protein, and a third open reading frame encoding the CEBPA protein.

[0014] In some embodiments, the one or more processed polynucleotides comprise a first polycistronic polynucleotide, which comprises the following open reading frames, ordered in the 5' to 3' direction of the first polycistronic polynucleotide: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the CEBPA protein, and a third open reading frame encoding the FLI1 protein.

[0015] In some embodiments, one or more processed polynucleotides comprise open reading frames encoding one or more of a MEF2C protein, a CEBPB protein, and an IRF8 protein.

[0016] In some embodiments, one or more processed polynucleotides comprise open reading frames encoding two or more of a MEF2C protein, a CEBPB protein, and an IRF8 protein.

[0017] In some embodiments, one or more processed polynucleotides comprise open reading frames encoding a MEF2C protein, a CEBPB protein, and an IRF8 protein.

[0018] In some embodiments, the one or more processed polynucleotides comprise a second polycistronic polynucleotide, which comprises the following open reading frames in the 5' to 3' direction of the second polycistronic polynucleotide: a first open reading frame encoding the MEF2C protein, a second open reading frame encoding the CEBPB protein, and a third open reading frame encoding the IRF8 protein.

[0019] In some embodiments, the one or more processed polynucleotides comprise a second polycistronic polynucleotide, which comprises the following open reading frames in the 5' to 3' direction of the second polycistronic polynucleotide: a first open reading frame encoding the MEF2C protein, a second open reading frame encoding the IRF8 protein, and a third open reading frame encoding the CEBPB protein.

[0020] In some embodiments, one or more processed polynucleotides include: a first polycistronic polynucleotide, which optionally includes from 5′ to 3′: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the CEBPA protein, and a third open reading frame encoding the FLI1 protein; and a second polycistronic polynucleotide, which optionally includes from 5′ to 3′: a first open reading frame encoding the MEF2C protein, a second open reading frame encoding the CEBPB protein, and a third open reading frame encoding the IRF8 protein.

[0021] In some embodiments, more than one open reading frame of the more than one processed polynucleotide is operably linked to a heterologous promoter.

[0022] In some embodiments, the heterologous promoter is an inducible promoter.

[0023] In some embodiments, the inducible promoter is a chemically inducible promoter.

[0024] Other aspects provide pluripotent stem cells (PSCs), comprising: SPI1 protein, FLI1 protein, and CEBPA protein, wherein the proteins are overexpressed.

[0025] In some embodiments, the PSC further comprises one or more of a MEF2C protein, a CEBPB protein, and an IRF8 protein, wherein one or more of the MEF2C protein, the CEBPB protein, and the IRF8 protein are overexpressed.

[0026] In some embodiments, the PSC further comprises two or more of a MEF2C protein, a CEBPB protein, and an IRF8 protein, wherein two or more of the MEF2C protein, the CEBPB protein, and the IRF8 protein are overexpressed.

[0027] In some embodiments, the PSC further comprises a MEF2C protein, a CEBPB protein, and an IRF8 protein, wherein the MEF2C protein, the CEBPB protein, and the IRF8 protein are overexpressed.

[0028] In some embodiments, the PSC is a human PSC.

[0029] In some embodiments, the PSC is an induced PSC (iPSC).

[0030] Certain aspects provide a composition comprising: a population of PSCs of any of the above paragraphs.

[0031] Other aspects provide methods, which include: culturing a population of pluripotent stem cells (PSCs) in a culture medium to produce an expanded population of PSCs; and expressing SPI1 protein, CEBPA protein, and FLI1 protein in the expanded population of PSCs to produce a population of microglia-like cells.

[0032] In some embodiments, the PSCs of the expanded population comprise more than one processed polynucleotide comprising an open reading frame encoding a SPI1 protein, a FLI1 protein, and a CEBPA protein.

[0033] In some embodiments, the one or more processed polynucleotides comprise a first polycistronic polynucleotide comprising: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the FLI1 protein, and a third open reading frame encoding the CEBPA protein.

[0034] In some embodiments, the one or more processed polynucleotides comprise a first polycistronic polynucleotide, which comprises the following open reading frames, ordered in the 5' to 3' direction of the first polycistronic polynucleotide: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the FLI1 protein, and a third open reading frame encoding the CEBPA protein.

[0035] In some embodiments, the one or more processed polynucleotides comprise a first polycistronic polynucleotide, which comprises the following open reading frames, ordered in the 5' to 3' direction of the first polycistronic polynucleotide: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the CEBPA protein, and a third open reading frame encoding the FLI1 protein.

[0036] In some embodiments, the method further comprises expressing one or more of a MEF2C protein, a CEBPB protein, and an IRF8 protein in the PSCs of the expanded population.

[0037] In some embodiments, the one or more processed polynucleotides further comprise an open reading frame encoding one or more of a MEF2C protein, a CEBPB protein, and an IRF8 protein.

[0038] In some embodiments, the method further comprises expressing two or more of a MEF2C protein, a CEBPB protein, and an IRF8 protein in the PSCs of the expanded population.

[0039] In some embodiments, the one or more processed polynucleotides further comprise open reading frames encoding two or more of a MEF2C protein, a CEBPB protein, and an IRF8 protein.

[0040] In some embodiments, the method further comprises expressing MEF2C protein, CEBPB protein, and IRF8 protein in the PSCs of the expanded population.

[0041] In some embodiments, the one or more processed polynucleotides further comprise open reading frames encoding MEF2C protein, CEBPB protein, and IRF8 protein.

[0042] In some embodiments, the one or more processed polynucleotides further comprise a second polycistronic polynucleotide, which comprises the following open reading frames in the 5' to 3' direction of the second polycistronic polynucleotide: a first open reading frame encoding the MEF2C protein, a second open reading frame encoding the CEBPB protein, and a third open reading frame encoding the IRF8 protein.

[0043] In some embodiments, the one or more processed polynucleotides further comprise a second polycistronic polynucleotide, which comprises the following open reading frames in the 5' to 3' direction of the second polycistronic polynucleotide: a first open reading frame encoding the MEF2C protein, a second open reading frame encoding the IRF8 protein, and a third open reading frame encoding the CEBPB protein.

[0044] In some embodiments, the PSC of the expanded population comprises: a first polycistronic polynucleotide, which optionally comprises from 5' to 3': a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the CEBPA protein, and a third open reading frame encoding the FLI1 protein; and a second polycistronic polynucleotide, which optionally comprises from 5' to 3': a first open reading frame encoding the MEF2C protein, a second open reading frame encoding the CEBPB protein, and a third open reading frame encoding the IRF8 protein.

[0045] In some embodiments, more than one open reading frame of the more than one processed polynucleotide is operably linked to a heterologous promoter.

[0046] In some embodiments, the heterologous promoter is an inducible promoter.

[0047] In some embodiments, the PSC population comprises 1×10 2 ~1×10 7 PSCs.

[0048] In some embodiments, the population of PSCs is cultured for about 2-5 days.

[0049] In some embodiments, the population of PSCs is cultured for about 4 days.

[0050] In some embodiments, the microglia-like cells of the microglia-like cell population are CD11b + 、CX3CR1 + 、ITGAM + 、P2RY12 + 、TMEM119 + and / or TREM2 + .

[0051] In some embodiments, the microglia-like cells of the microglia-like cell population are TRA-1-60 - and / or POU5F1 - .

[0052] Some aspects provide a method comprising: (a) delivering one or more processed polynucleotides to pluripotent stem cells (PSCs), wherein the processed polynucleotides comprise an inducible promoter operably linked to one or more open reading frames encoding SPI1 protein, CEBPA protein, and FLI1 protein, optionally wherein the one or more processed polynucleotides further comprise an inducible promoter operably linked to one or more open reading frames encoding MEF2C protein, CEBPB protein, and / or IRF8 protein; (b) culturing the PSCs of the expanded population in an induction medium comprising an induction agent to produce CD11b + 、CX3CR1 + 、ITGAM + 、P2RY12 + 、TMEM119 + TREM2 + TRA-1-60 - and / or POU5F1 - Microglia-like cells.

[0053] Other aspects provide a method comprising: (a) delivering one or more processed polynucleotides to pluripotent stem cells (PSCs), wherein the processed polynucleotides comprise an inducible promoter operably linked to one or more open reading frames encoding SPI1 protein, CEBPA protein and FLI1 protein, and optionally wherein the one or more processed polynucleotides further comprise an inducible promoter operably linked to one or more open reading frames encoding MEF2C protein, CEBPB protein and / or IRF8 protein; (b) seeding the PSCs in a feeder-free, serum-free culture medium, and optionally culturing the PSCs for about 1 to about 24 hours; (c) culturing the PSCs of (b) in an induction medium comprising an induction agent to produce CD11b + 、CX3CR1 + 、ITGAM + 、P2RY12 + 、TMEM119 + TREM2 + TRA-1-60 - and / or POU5F1 - Microglia-like cells.

[0054] In some embodiments, the method further comprises delivering to the PSC (i) a first polycistronic polynucleotide comprising a first inducible promoter operably linked to an open reading frame encoding SPI1, an open reading frame encoding CEBPA, and an open reading frame encoding FLI1, and (ii) a second polycistronic polynucleotide comprising a second inducible promoter operably linked to an open reading frame encoding MEF2C, an open reading frame encoding CEBPB, and an open reading frame encoding IRF8.

[0055] In some embodiments, the first and / or second polycistronic polynucleotide is a transposon, and delivering further comprises delivering a transposase to the PSC.

[0056] In some embodiments, the first and / or second inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

[0057] In some embodiments, the feeder-free, serum-free medium of (b) comprises a soluble basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

[0058] In some embodiments, the solubilized basement membrane preparation includes extracellular matrix (ECM) proteins and growth factors.

[0059] In some embodiments, the ECM protein is selected from laminin, type IV collagen, heparan sulfate proteoglycans, and entactin.

[0060] In some embodiments, the feeder-free, serum-free culture medium further comprises a growth factor selected from recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ).

[0061] In some embodiments, the PSCs of the expanded population of (c) are approximately 20,000 cells / cm 2 ~60,000 cells / cm 2 Density culture.

[0062] In some embodiments, the feeder-free, serum-free culture medium of (b) further comprises a small molecule ROCK inhibitor.

[0063] In some embodiments, the inducing agent comprises doxycycline.

[0064] In some embodiments, the PSCs are cultured in the induction medium for about 72 to about 96 hours.

[0065] Certain aspects provide microglia-like cells produced by the method of any of the above paragraphs.

Brief Description of the Figures

[0067] The drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component illustrated in the various figures is represented by a like numeral. For clarity, not every component may be labeled in every drawing. In the drawings:

[0068]

【 Figures 1A to 1I The first iteration of TF screening identified initial microglial differentiation factors. Figure 1A ) The first workflow for incorporating TF screening. Figure 1B ) Flow cytometric analysis of stem cell (TRA-1-60) and microglial (P2RY12, CD11b, CX3CR1) proteins in the PGP1+40TF pool before and after Dox induction. Figure 1C ) scRNA-seq sequencing was performed on cells with low TRA-1-60 expression in the Dox+ group. ( Figure 1D ) Aggregation of two independently transfected and differentiated PGP1 iPSC pools. Figure 1E ) Expression of microglial (ITGAM, CX3CR1, TMEM119, P2RY12, TREM2) and stem cell-added (POU5F1) genes in scRNA-seq. Figure 1F ) Primer design for co-amplification of TFs and cellular barcodes in the 10×Genomics 3′ workflow. Figure 1G ) Number of TFs per cell counted from the normalized and binarized TF expression matrix. ( Figure 1H )Ranking of 40 TFs after Wilcoxon rank sum test, two test groups with or without microglial gene expression. Asterisks highlight the top ranked TFs. Fig. 1I ) Flow cytometric validation of the top ranked TFs for inducing protein expression in microglia.

[0069]

【 Figures 2A to 2J A second iteration based on the first iteration identified additional TFs for microglia differentiation. Figure 2A ) The workflow for the second merged TF screening. Figure 2B ) Polycistronic cassette design for dual-drug selection to achieve 3+X TF screening. Figure 2C) Normalized mRNA expression from polycistronic cassettes (SPI1, FLI1, CEBPA) and stem cells (POU5F1). (Fig. 2dD) TF barcode counts enable identification of stem cells (“No TF BC”), MG3.1-SFC, and cells with additional TFs (“SFC+X”). (Fig. 2e) Example histograms of TF barcode raw counts in individual cells. ( Figure 2F ) Number of TFs per cell counted from the normalized and binarized TF expression matrix. ( Figure 2G )Ranking of 42 TFs after Wilcoxon rank sum test, two test groups with or without microglial gene expression. Asterisks highlight the top ranked TFs. Grey highlights the SFC polycistronic cassette. ( Figure 2H ) Flow cytometry validation of the top ranked TFs for improving microglial protein expression. ( Fig.2I ) The polycistronic cassette design is suitable for different TF sequences. Figure 2J ) Flow cytometric analysis of different permutations of 6-TF formulations compared to MG3.1-SFC.

[0070]

Figures 3A to 3J

[0071]

Figures 4A to 4E

[0073] The ability to generate human cell types from stem cells provides a critical tool for basic research and therapeutic development, especially when the desired cell type is not easily obtained from a human biopsy. The broader and deeper knowledge of developmental biology accumulated over the years has driven the discovery and application of many protocols for creating specific cell types from iPSCs, some of which have entered clinical trials for the treatment of devastating diseases such as age-related macular degeneration and type 1 diabetes, while others are routinely used in the laboratory to study disease mechanisms and test drugs. Recent global efforts to establish HCAs have not only added more knowledge about human development and disease, but also proposed many clear targets for cell fate engineering. Combined with the development of gene library construction, high-throughput screening and sequencing technologies, now is the best time to study how to engineer cell fate in a systematic and multiplexed manner.

[0074] The data presented here demonstrate the feasibility of cell fate engineering using iterative genetic library screening and high-throughput scRNA-seq. Using previously unavailable TF-driven protocols for microglial differentiation, two complete design-screen-validation iterations were performed, and SPI1, CEBPA, FLI1, MEF2C, CEBPB, and IRF8 were identified as recipes that drive microglia differentiation from hiPSCs in, for example, about 4 days. Characterization of these transcription factor-induced microglia-like cells (TFiMGLs) showed that they differentiate rapidly in about 4 days and have transcriptomic and functional similarities to primary human microglia.

[0075] Microglia-like cells

[0076] Certain aspects of the present disclosure provide microglia-like cells and methods for producing such cells. Microglia are innate immune cells in the central nervous system (CNS) and have two main roles in the brain that are common to granulocytes and monocytic innate immune cells in all mammalian organs: they resist and engulf viruses, bacteria and other foreign invaders, and remove cell debris to promote wound repair. Innate immune cells are sparsely distributed in the liver, lungs and skin. In contrast, microglia occupy all areas of the CNS in a tiled pattern. Because the entire brain of an adult mammal is occupied by microglia, it is clear that they have brain-specific functions. Microglia originate from unique stem cells in the yolk sac. Microglial progenitor cells colonize the brain during early fetal development and initially have an amoeba shape. In order to ensure that appropriate neuronal connections are established during brain development, the CNS overproduces neurons, and developing neurons compete for connections. Neurons that lose this competition die; microglia do not kill these neurons, but they recognize their dysfunction or degeneration and remove them.

[0077] As the prenatal brain develops, individual microglia establish microdomains and extend processes symmetrically. Cell-autonomous contact inhibition helps establish and maintain the CNS network of microglia. Branching microglia comprise 5% to 20% of adult CNS glial cells. They are more abundant in gray matter than in white matter, and phylogenetically newer regions of the CNS (cerebral cortex, hippocampus) have more microglia than older regions (cerebellum, brainstem). Regional differences in microglia number and shape suggest that microglia distribution and morphology are regulated by the local environment and that microglia play a role in tissue homeostasis. Although many aspects of this homeostasis remain to be elucidated, microglia respond rapidly and dramatically to all forms of brain pathology.

[0078] Microglia have two major functional aspects: immune defense and CNS maintenance. As immune cells, they serve as sentinels, detecting the first signs of pathogen invasion or tissue damage in this vulnerable immune-privileged site that is actively protected by the brain-blood barrier. However, under inflammatory conditions of active immune responses, microglia must also moderate potential damage to the CNS and support tissue repair and remodeling. Perhaps unsurprisingly, dysregulated microglial activation and microglia-induced inflammation are observed in nearly all brain pathologies; emerging evidence suggests that microglia exert direct effects on neurons that contribute to disease progression.

[0079] Microglia express many different biomarkers that can be used to distinguish microglia and microglia-like cells from other cell types. For example, microglia are usually positive for CD11b, CX3CR1, ITGAM, P2RY12, TMEM119 and / or TREM2 (i.e., they express CD11b, CX3CR1, ITGAM, P2RY12, TMEM119 and / or TREM2). Microglia are usually negative for TRA-1-60 and / or POU5F1 (i.e., they do not express detectable levels of TRA-1-60 and / or POU5F1). Therefore, in some embodiments, the microglia-like cells produced by the methods provided herein are CD11b + 、CX3CR1 + 、ITGAM + 、P2RY12 + 、TMEM119 + and / or TREM2 + Microglia-like cells and / or TRA-1-60 - and / or POU5F1 - Microglia-like cells (i.e., cells that express CD11b, CX3CR1, ITGAM, P2RY12, TMEM119 and / or TREM2 proteins but do not express detectable levels of TRA-1-60 and / or POU5F1 proteins).

[0080] Microglia-like cells have other features that distinguish them from non-microglia-like cells, including but not limited to secretion of cytokines (TNFα, IL-6, IL-10) upon stimulation (IFNγ, LPS), phagocytosis of disease (bacterial particles, amyloid β) or other brain-associated (synaptosomal) agents, and response to damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), signals or molecular motifs released from damaged or dying cells (i.e., ADP) that are conserved in the microbiome.

[0081] In some embodiments, at least 50% of the cells in the microglia-like cell population are CD11b + For example, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the cells in a population of microglia-like cells may be CD11b + In some embodiments, about 50% to about 80% of the cells in a population of microglia-like cells are CD11b + For example, about 50% to about 70%, about 60% to about 80%, or about 60% to about 70% of the cells in a population of microglia-like cells may be CD11b + .

[0082] In some embodiments, at least 70% of the cells in the microglia-like cell population are P2RY12 + For example, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the cells in a population of microglia-like cells may be P2RY12 + In some embodiments, about 80% to about 95% of the cells in the microglia-like cell population are P2RY12 + For example, about 70% to about 95%, about 75% to about 95%, or about 80% to about 95% of the cells in a population of microglia-like cells may be P2RY12 + .

[0083] In some embodiments, at least 5% of the cells in the microglia-like cell population are CX3CR1 + For example, at least 5%, at least 10%, or at least 15% of the cells in a population of microglia-like cells may be CX3CR1 + In some embodiments, about 5% to about 25% of the cells in the microglia-like cell population are CX3CR1 + For example, about 5% to about 20%, about 10% to about 20%, or about 15% to about 20% of the cells in a population of microglia-like cells may be CX3CR1 + .

[0084]

Pluripotent stem cells

[0085] The microglia-like cells provided herein are differentiated from pluripotent stem cells. Pluripotent stem cells are cells that have the ability to self-renew by dividing and develop into the three major germ cell layers of the early embryo (e.g., ectoderm, endoderm, and mesoderm), and thus into all cells of the adult body, excluding extraembryonic tissues such as the placenta (Shi et al., 2017).

[0086] Non-limiting examples of pluripotent stem cells include induced pluripotent cells (iPSCs), "true" embryonic stem cells (ESCs) derived from embryos, embryonic stem cells made by somatic cell nuclear transplantation (ntESCs), and embryonic stem cells from unfertilized eggs (parthenogenetic embryonic stem cells, or pESCs). In some embodiments, the pluripotent cells are human pluripotent cells.

[0087] In some embodiments, the pluripotent stem cell is an embryonic stem cell, such as a human embryonic stem cell. "Embryonic stem cell" is a general term for pluripotent stem cells made from embryos or eggs, rather than cells that have been genetically reprogrammed from the body. As used herein, "ESC" includes true ESC, ntESC, and pESC.

[0088] In other embodiments, the pluripotent stem cells are induced pluripotent stem cells, such as human induced pluripotent stem cells. iPSCs may be derived from skin or blood cells that have been reprogrammed back to an embryonic-like pluripotent state, enabling the development of an unlimited source of any type of human cell.

[0089] Certain aspects of the present disclosure provide a PSC comprising: a protein selected from SPI1, CEBPA, FLI1, MEF2C, CEBPB and IRF8, wherein the protein is expressed or overexpressed. In some embodiments, the expression level of the protein is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50% or at least 100% higher than the control level. In some embodiments, the control level is the endogenous level of the protein, such as in naturally occurring pluripotent stem cells. In some embodiments, the PSC comprises SPI1. In some embodiments, the PSC expresses or overexpresses SPI1. In some embodiments, the PSC comprises CEBPA. In some embodiments, the PSC expresses or overexpresses CEBPA. In some embodiments, the PSC comprises FLI1. In some embodiments, the PSC expresses or overexpresses FLI1. In some embodiments, the PSC comprises MEF2C. In some embodiments, the PSC expresses or overexpresses MEF2C. In some embodiments, the PSC comprises CEBPB. In some embodiments, the PSC expresses or overexpresses CEBPB. In some embodiments, the PSC comprises IRF8. In some embodiments, the PSC expresses or overexpresses IRF8.

[0090] The data provided herein show that the combined expression of SPI1, FLI1 and CEBPA results in 37% of the cell population being CD11b+ and 6% being P2RY12+. Other data provided herein show that the combined expression of SPI1, FLI1, CEBPA, MEF2C, CEBPB and IRF8 results in 66% of the cell population being CD11b+, 93% being P2RY12+, and 16% being CX3CR1+ on the 4th day after induction of expression of transcription factors. In some embodiments, the PSC comprises SPI1, FLI1 and CEBPA. In some embodiments, the PSC expresses or overexpresses SPI1, FLI1 and CEBPA. In some embodiments, the PSC comprises SPI1, FLI1, CEBPA and one or more of MEF2C, CEBPB and IRF8. In some embodiments, the PSC comprises SPI1, FLI1, CEBPA and two or more of MEF2C, CEBPB and IRF8. In some embodiments, the PSC comprises SPI1, FLI1, CEBPA, MEF2C, CEBPB, and IRF8. In some embodiments, the PSC expresses or overexpresses SPI1, FLI1, CEBPA, and one or more of MEF2C, CEBPB, and IRF8. In some embodiments, the PSC expresses or overexpresses SPI1, FLI1, CEBPA, and two or more of MEF2C, CEBPB, and IRF8. In some embodiments, the PSC expresses or overexpresses SPI1, FLI1, CEBPA, MEF2C, CEBPB, and IRF8.

[0091]

Transcription factors

[0092] The microglia-like cells provided herein are differentiated from pluripotent stem cells by expressing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) transcription factors (i.e., proteins that control transcription rates) in some embodiments. Differentiation is the process by which uncommitted cells or partially committed cells determine a specific cell fate. Various aspects of the present disclosure relate to the differentiation of uncommitted pluripotent stem cells into microglia-like cell fates.

[0093] In some embodiments, the transcription factor is selected from SPI1, CEBPA, FLI1, MEF2C, CEBPB and IRF8. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are processed to express or overexpress SPI1. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are processed to express or overexpress CEBPA. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are processed to express or overexpress FLI1. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are processed to express or overexpress MEF2C. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are processed to express or overexpress CEBPB. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are processed to express or overexpress IRF8.

[0094] In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are processed to express or overexpress SPI1, CEBPA and FLI1. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are processed to express or overexpress SPI1, CEBPA and FLI1 and one or more of MEF2C, CEBPB and IRF8. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are processed to express or overexpress SPI1, CEBPA and FLI1 and two or more of MEF2C, CEBPB and IRF8. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are processed to express or overexpress SPI1, CEBPA, FLI1, MEF2C, CEBPB and IRF8.

[0095] A cell "expresses" a particular protein if the level of the protein in the cell is detectable (e.g., using a known protein assay). A cell "overexpresses" a protein (e.g., a processed polynucleotide encoding the protein) if the level of the protein is higher (e.g., at least 5%, at least 10%, or at least 20% higher) than the level of the protein expressed by an endogenous, native polynucleotide encoding the protein.

[0096]

Processed polynucleotides and polypeptides

[0097] The pluripotent stem cells disclosed herein, in some embodiments, comprise processed polynucleotides. A processed polynucleotide is a nucleic acid that does not exist in nature (e.g., at least two nucleotides covalently linked together, in some cases, containing a phosphodiester bond, referred to as a phosphodiester backbone). Processed polynucleotides include recombinant nucleic acids and synthetic nucleic acids. Recombinant nucleic acids are molecules constructed by connecting nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or a combination thereof) from two different organisms (e.g., humans and mice). Synthetic nucleic acids are molecules that are amplified or chemically or otherwise synthesized. Synthetic nucleic acids include those that have been chemically modified or otherwise modified but can base pair (bind) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include molecules produced by any of the above replications.

[0098] The processed polynucleotides can comprise DNA (e.g., genomic DNA, cDNA, or a combination of genomic DNA and cDNA), RNA, or a hybrid molecule, for example, in which the nucleic acid comprises any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural), and any combination of two or more bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine.

[0099] In some embodiments, the polynucleotide is complementary DNA (cDNA).cDNA is synthesized from a single-stranded RNA (eg, messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by reverse transcriptase.

[0100] The processed polynucleotides of the present disclosure can be generated using standard molecular biology methods (see, for example, Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the nucleic acid is generated using GIBSON Generated by cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009; and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). Three enzyme activities are typically used in a single tube reaction: 5' exonuclease, 3' extension activity of DNA polymerase, and DNA ligase activity. The 5' exonuclease activity chews the 5' end sequence and exposes the complementary sequence for annealing. The polymerase activity then fills the gap on the annealing domain. The DNA ligase then seals the gap and covalently connects the DNA fragments together. The overlapping sequences of adjacent fragments are much longer than those used in Golden Gate Assembly, thus resulting in a higher percentage of correct assembly. Other methods for producing processed polynucleotides can be used in accordance with the present disclosure.

[0101] In some embodiments, the processed polynucleotide comprises a promoter operably connected to an open reading frame. A promoter is a nucleotide sequence (e.g., ATG) to which RNA polymerase binds to initial transcription. A promoter is typically located directly upstream (5' end) of the transcription start site. In some embodiments, a promoter is a heterologous promoter. A heterologous promoter is not naturally associated with the open reading frame to which it is operably connected.

[0102] In some embodiments, the promoter is an inducible promoter. For example, an inducible promoter can be regulated by chemical reagents, temperature or light in vivo. For example, an inducible promoter can control gene expression in time and / or space. Inducible promoters used according to the present disclosure include any inducible promoters described herein or known to those of ordinary skill in the art. Examples of inducible promoters include, but are not limited to, chemically / biochemically regulated and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, including tetracycline repressor protein (tetR), tetracycline operator sequence (tetO), and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (for example, promoters based on rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptor, and steroid / retinoid / thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters from metallothionein (proteins that bind and sequester metal ions) genes derived from yeast, mouse, and humans), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene, or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light-responsive promoters from plant cells). In some embodiments, the inducible promoter is a tetracycline inducible promoter. In some embodiments, the inducible promoter is a doxycycline inducible promoter. In other embodiments, the promoter is a constitutive promoter (active in vivo, not regulated).

[0103] An open reading frame is a continuous stretch of codons that begins with a start codon (e.g., ATG) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a polypeptide, e.g., a protein. An open reading frame is operably linked to a promoter if the promoter regulates the transcription of the open reading frame.

[0104] Vectors for delivering processed polynucleotides include minicircles, plasmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes. Transposon-based systems, such as piggyBac TM systems (e.g., Chen et al., Nature Communications. 2020; 11(1): 3446), are also considered here.

[0105] In some embodiments, the pluripotent stem cell comprises a processed polynucleotide comprising an open reading frame encoding a protein selected from SPI1, CEBPA, FLI1, MEF2C, CEBPB, and IRF8. In some embodiments, the processed polynucleotide comprises an open reading frame encoding SPI1. In some embodiments, the processed polynucleotide comprises an open reading frame encoding CEBPA. In some embodiments, the processed polynucleotide comprises an open reading frame encoding FLI1. In some embodiments, the processed polynucleotide comprises an open reading frame encoding MEF2C. In some embodiments, the processed polynucleotide comprises an open reading frame encoding CEBPB. In some embodiments, the processed polynucleotide comprises an open reading frame encoding IRF8.

[0106] In some embodiments, the processed polynucleotide encoding an open reading frame encoding SPI1 (also known as transcription factor PU.1) (eg, UniprotKB Accession No. P17947) encodes a protein comprising the following sequence:

[0107] MLQACKMEGF PLVPPPSEDL VPYDTDLYQR QTHEYYPYLSSDGESHSDHY WDFHPHHVHS

[0108] EFESFAENNF TELQSVQPPQ LQQLYRHMEL EQMHVLDTPM VPPHPSLGHQ VSYLPRMCLQ

[0109] YPSLSPAQPSSDEEEGERQS PPLEVSDGEA DGLEPGPGLL PGETGSKKKI RLYQFLLDLL

[0110] RSGDMKDSIW WVDKDKGTFQ FSSKHKEALA HRWGIQKGNR KKMTYQKMARALRNYGKTGEVKKVKKKLTY QFSGEVLGRG GLAERRHPPH(SEQ ID NO: 1)

[0111] In some embodiments, the processed polynucleotide encoding an open reading frame encoding CCAAT / enhancer binding protein alpha (CEBPA) (e.g., UniprotKB Accession No. P49715) encodes a protein comprising the following sequence:

[0112]

[0113] In some embodiments, the processed polynucleotide encoding an open reading frame encoding Friend Leukemia Integration 1 Transcription Factor (FLI1) (eg, UniprotKB Accession No. Q01543) encodes a protein comprising the following sequence:

[0114]

[0115] In some embodiments, the processed polynucleotide encoding an open reading frame encoding myocyte-specific enhancer factor 2C (MEF2C) (eg, UniprotKB Accession No. Q06413) encodes a protein comprising the following sequence:

[0116]

[0117] In some embodiments, the processed polynucleotide encoding an open reading frame encoding CCAAT / enhancer binding protein beta (CEBPB) (e.g., UniprotKB Accession No. P17676) encodes a protein comprising the following sequence:

[0118]

[0119] In some embodiments, the processed polynucleotide encoding an open reading frame encoding interferon regulatory factor 8 (IRF8) (e.g., UniprotKB Accession No. Q02556) encodes a protein comprising the following sequence:

[0120]

[0121] The number of copies of the processed polynucleotide delivered to the PSC may vary. In some embodiments, the PSC comprises 1 to 20 copies of the processed polynucleotide. For example, the PSC may comprise 1 to 15, 1 to 10, 2 to 10, 2 to 15, 2 to 10, 5 to 20, 5 to 15, or 5 to 10 copies of the processed polynucleotide. In some embodiments, the PSC comprises 8 to 10 copies of the processed polynucleotide. More than 20 copies are also contemplated herein.

[0122] In some embodiments, the processed polynucleotide encodes a variant of SPI1, CEBPA, FLI1, MEF2C, CEBPB or IRF8 protein. Protein variants are proteins (including full-length proteins and peptides) that are different in amino acid sequence relative to wild-type, native or reference amino acid sequences. Compared to wild-type, native or reference amino acid sequences, protein variants may have more than one substitution, deletion and / or insertion at certain positions in their amino acid sequences. Typically, protein variants have at least 50% identity with wild-type, native or reference sequences. In some embodiments, protein variants have at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with wild-type, native or reference sequences.

[0123] Protein variants may contain amino acid changes that confer any of a number of desirable properties (e.g., enhancing its expression and / or improving its stability). Protein variants can be prepared using conventional mutagenesis techniques and analyzed as appropriate to determine whether they have the desired properties. Assays for determining protein expression levels (including protein variants) are well-known in the art. The stability of protein variants can be measured by measuring thermal stability or stability after urea denaturation, or can be measured using methods such as computer prediction. Methods for such experiments and computer determinations are known in the art.

[0124] In some embodiments, the processed polynucleotide comprises an open reading frame encoding an amino acid sequence comprising any one of the sequences provided herein, or a protein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of any one of the sequences provided herein. For example, see SEQ ID NOs: 1 to 6.

[0125] "Identity" refers to the relationship between two or three or more sequences (e.g., amino acid sequences or nucleotide sequences), determined by comparing the sequences to each other. Identity also refers to the degree of sequence relatedness between or among sequences, as determined by the number of matches between or among amino acid chains (polypeptides) or nucleotide chains (polynucleotides). Identity is a measure of the percentage of identical matches between the smaller of two or more sequences, with gapped alignments (if any) addressed by a particular mathematical model or computer program (e.g., an "algorithm"). The identification of related polypeptides and polynucleotides can be readily calculated by known methods. "Percent (%) identity" as applied to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid or nucleic acid residues) in a candidate (first) polypeptide or polynucleotide sequence that are identical to the residues (amino acid or nucleic acid residues) in a second polypeptide or polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage identity.

[0126] Methods and computer programs for comparison are well known in the art. It is understood that identity depends on the calculation of the identity percentage, but due to the gaps and penalties introduced in the calculation, its value may vary. Generally, variants of specific polynucleotides or polypeptides have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity with the sequence described herein and known to those skilled in the art and the specific wild type, natural or reference sequence. Such comparison tools include, but are not limited to, tools of the BLAST suite (Altschul, SF, et al. Nucleic Acids Res. 1997; 25: 3389-3402); and algorithms based on the Smith-Waterman algorithm (Smith, TF & Waterman, MSJ Mol. Biol. 1981; 147: 195-197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CDJ Mol. Biol. 1920; 48: 443-453). In addition, a fast optimal global sequence alignment algorithm (FOGSAA) has been developed, which is said to generate global alignments of nucleotide and protein sequences faster than other optimal global alignment methods (including the Needleman-Wunsch algorithm).

[0127] Therefore, polynucleotides and polypeptides contain substitutions, insertions and / or deletions (e.g., indels), as well as covalent modifications of wild-type, native or reference sequences, e.g., polypeptide (e.g., protein) sequences disclosed herein, are included within the scope of the present disclosure. For example, sequence tags or amino acids (e.g., one or more lysines) can be added to a polypeptide sequence (e.g., at the N-terminus and / or C-terminus). Sequence tags can be used for peptide detection, purification and / or localization. Lysine can be used to increase the solubility of a peptide or to allow biotinylation. Alternatively, amino acid residues located in the N-terminal and / or C-terminal regions of a protein amino acid sequence can be selected for deletion, thereby providing a truncated sequence.

[0128] As those skilled in the art will appreciate, protein fragments, functional protein domains and homologous proteins are also considered to be within the scope of proteins provided herein. For example, any protein fragment of a wild-type, native sequence or reference sequence is provided herein (representing that the polypeptide sequence is at least shorter than the wild-type, native sequence or reference sequence but is otherwise identical), provided that the fragment retains its wild-type function. In addition to protein variants identical to wild-type, natural or reference proteins but truncated, in some embodiments, the protein also includes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations (e.g., replacements, insertions and / or deletions), as shown in any sequence provided herein or quoted. The length of protein variants ranges from about 4, 6 or 8 amino acids to full-length proteins.

[0129]

Polycistronic polynucleotide

[0130] In some embodiments, the pluripotent stem cell comprises more than one polycistronic polynucleotide. The polycistronic polynucleotide encodes multiple proteins on a single polynucleotide molecule. In some embodiments, the pluripotent stem cell comprises a processed polycistronic polynucleotide comprising an open reading frame encoding SPI1, an open reading frame encoding CEBPA, and an open reading frame encoding FLI1. In some embodiments, the pluripotent stem cell comprises a processed polycistronic polynucleotide comprising an open reading frame encoding MEF2C, an open reading frame encoding CEBPB, and an open reading frame encoding IRF8.

[0131] The coding order of the open reading frames on the polycistronic polynucleotide affects the expression level of the protein. Generally, the open reading frames placed in the upstream (5') position are expressed at the highest level relative to other open reading frames. In some embodiments, the polycistronic polynucleotide 5' to 3' (in the following order) comprises: an open reading frame encoding SPI1, an open reading frame encoding CEBPA, and an open reading frame encoding FLI1. In other embodiments, the polycistronic polynucleotide 5' to 3' (in the following order) comprises: an open reading frame encoding SPI1, an open reading frame encoding FLI1, and an open reading frame encoding CEBPA. In some embodiments, the polycistronic polynucleotide 5' to 3' (in the following order) comprises: an open reading frame encoding MEF2C, an open reading frame encoding CEBPB, and an open reading frame encoding IRF8. In other embodiments, the polycistronic polynucleotide 5' to 3' (in the following order) comprises: an open reading frame encoding MEF2C, an open reading frame encoding IRF8, and an open reading frame encoding CEBPB.

[0132] In some embodiments, the open reading frame is separated from each open reading frame by a sequence encoding a self-cleaving peptide, such as a 2A self-cleaving peptide (e.g., T2A, P2A, E2A, and F2A). In some embodiments, the open reading frame is separated from each reading frame by an internal ribosome entry site (IRES).

[0133] Method for generating microglia-like cells

[0134] The method for producing microglia-like cells provided herein, in some aspects, comprises culturing a population of pluripotent stem cells (PSCs) in a culture medium to produce an expanded population of PSCs; and expressing a protein selected from SPI1, CEBPA, FLI1, MEF2C, CEBPB and IRF8 (e.g., SPI1, CEBPA and FLI1; or SPI1, CEBPA, FLI1, MEF2C, CEBPB and IRF8) in the expanded population of PSCs to produce microglia-like cells.

[0135] In some embodiments, the PSC of the amplified population comprises a processed polynucleotide comprising an open reading frame encoding SPI1. In some embodiments, the PSC of the amplified population comprises a processed polynucleotide comprising an open reading frame encoding CEBPA. In some embodiments, the PSC of the amplified population comprises a processed polynucleotide comprising an open reading frame encoding FLI1. In some embodiments, the PSC of the amplified population comprises a processed polynucleotide comprising an open reading frame encoding MEF2C. In some embodiments, the PSC of the amplified population comprises a processed polynucleotide comprising an open reading frame encoding CEBPB. In some embodiments, the PSC of the amplified population comprises a processed polynucleotide comprising an open reading frame encoding IRF8.

[0136] In some embodiments, the PSC of the expanded population comprises a processed polycistronic polynucleotide comprising an open reading frame encoding SPI1, an open reading frame encoding CEBPA, and an open reading frame encoding FLI1. In some embodiments, the PSC of the expanded population also includes a processed polycistronic polynucleotide comprising an open reading frame encoding MEF2C, an open reading frame encoding CEBPB, and an open reading frame encoding IRF8.

[0137] In some embodiments, the open reading frame of the processed polynucleotide is operably linked to a heterologous promoter.

[0138] In some embodiments, the heterologous promoter is an inducible promoter, non-limiting examples of which are provided elsewhere herein.

[0139] The starting population contained approximately 1×10 2 ~1×10 10 , about 1×10 2 ~1×10 9 , about 1×10 2 ~1×10 8 or about 1×10 2 ~1×10 7 In some embodiments, the population comprises about 1×10 3 ~1×10 8 or about 1×10 3 ~1×10 7 In some embodiments, the population comprises about 1×10 4 ~1×10 7 or about 1×10 5 ~1×10 6In some embodiments, the population comprises about 1×10 1 PSCs, about 1×10 2 PSCs, about 1×10 3 PSCs, about 1×10 4 PSCs, about 1×10 5 PSCs, about 1×10 6 PSCs, about 1×10 7 PSCs, about 1×10 8 PSCs, about 1×10 9 PSCs or about 1×10 10 PSCs.

[0140] In some embodiments, the PSC population or expanded PSC population is cultured for about 4 to about 10 days, about 4 to about 9 days, about 4 to about 8 days, about 4 to about 7 days, about 4 to about 6 days, about 5 to about 10 days, about 5 to about 9 days, about 5 to about 8 days, about 5 to about 7 days, or about 5 to about 6 days. In some embodiments, the PSC population is cultured for about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0141] Some methods disclosed herein provide methods comprising (a) delivering one or more processed polynucleotides to PSCs, the polynucleotides comprising an inducible promoter operably linked to one or more open reading frames encoding a protein selected from SPI1, CEBPA, FLI1, MEF2C, CEBPB, and IRF8; (b) culturing the PSCs in a feeder-free, serum-free medium to generate a larger population of PSCs; (c) culturing the PSCs of the expanded population in an induction medium comprising an induction agent to produce CD11b + 、CX3CR1 + 、ITGAM + 、P2RY12 + 、TMEM119 + TREM2 + TRA-1-60 - and / or POU5F1 - Microglia-like cells.

[0142] In some embodiments, the PSC is cultured in a feeder-free, serum-free medium for about 6 to about 24 hours. For example, the PSC can be cultured in a feeder-free, serum-free medium for about 6 to about 12 hours. In some embodiments, the PSC is cultured in a feeder-free, serum-free medium for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.

[0143] In some embodiments, the expanded population of PSCs comprises at least 5×10 3 For example, an expanded population (e.g., upon induction) may include at least 1×10 4 , at least 1×10 5 , at least 1×10 6 or at least 1×10 7 In some embodiments, the expanded PSC population comprises about 5×10 3 PSC~about 1×10 7 PSCs.

[0144] In some embodiments, the PSCs of the expanded population are grown at about 10,000 cells / cm 2 ~ About 30,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 10,000 cells / cm 2 ~25,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 10,000 cells / cm 2 ~20,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 10,000 cells / cm 2 ~About 15,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 15,000 cells / cm 2 ~ About 30,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 15,000 cells / cm 2 ~25,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 15,000 cells / cm 2 ~20,000 cells / cm 2In some embodiments, the PSCs of the expanded population are cultured at a density of at least 10,000 / cm 2 , at least 15,000 / cm 2 , at least 20,000 / cm 2 , at least 25,000 / cm 2 or at least 30,000 / cm 2 Density culture.

[0145] In some embodiments, the PSC culture time of the expanded population is no more than 10 days, no more than 9 days, no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days or no more than 4 days. For example, the PSC of the expanded population can be cultured for about 4 to 10 days, about 4 to about 9 days, about 4 to about 8 days, about 4 to about 7 days, about 4 to about 6 days, about 5 to about 10 days, about 5 to about 9 days, about 5 to about 8 days, about 5 to about 7 days, or about 5 to about 6 days. In some embodiments, the PSC of the expanded population is cultured for about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or about 10 days.

[0146] In some embodiments, the PSC of the expanded population is cultured in the induction medium for about 36 to about 60 hours. For example, the PSC can be cultured in the induction medium for about 36 to 54 hours, about 36 to about 48 hours, about 42 to about 60 hours, about 42 to about 54 hours, about 42 to about 48 hours, about 48 to about 60 hours, or about 48 to about 54 hours. In some embodiments, the PSC is cultured in the induction medium for about 36 hours, about 42 hours, about 48 hours, about 54 hours, or about 60 hours.

[0147] Cultivation in the second induction medium includes, in some embodiments, several (one or more) changes of culture medium. For example, the induction medium can be removed and replaced with a new (fresh) second indicator medium every (approximately) 12 hours, every 24 hours, every 36 hours, or every 48 hours. In some embodiments, the induction medium is replaced every (approximately) 24 hours.

[0148]

Transfection method

[0149] The processed polynucleotides of the present disclosure can be delivered to PSCs using any one or more transfection methods, including chemical transfection methods, viral transduction methods, and electroporation.

[0150] In some embodiments, the processed polynucleotide is delivered on a vector. The vector is any vector, such as a virus or plasmid, for transferring the desired polynucleotide to a host cell (such as a PSC). In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is not a naturally occurring viral vector. The viral vector may be from adeno-associated virus (AAV), adenovirus, herpes simplex virus, lentivirus, retrovirus, varicella, smallpox virus, hepatitis B, cytomegalovirus, JC polyomavirus, BK polyomavirus, monkeypox virus, herpes zoster virus, Epstein-Barr virus, human herpes virus 7, Kaposi's sarcoma-associated herpes virus or human parvovirus B19. The present disclosure includes other viral vectors.

[0151] In some embodiments, the viral vector is an AAV vector. AAV is a small, non-enveloped virus that packages a single-stranded linear DNA genome of about 5 kb and has been adapted for use as a gene transfer vector (Samulski, RJ et al., Annu Rev Virol. 2014; 1 (1): 427-51). The coding region of AAV is flanked by inverted terminal repeats (ITRs), which act as the origin of DNA replication and as primary packaging signals (McLaughlin, SK et al. Virol. 1988; 62 (6): 1963-73; Hauswirth, WW et al., 1977; 78 (2): 488-99). Therefore, AAV vectors typically include ITR sequences. Both the positive and negative strands are equally well packaged into viral particles and are able to infect (Zhong, L et al. Mol Ther. 2008; 16 (2): 290-5; Zhou, X et al. Mol Ther. 2008; 16 (3): 494-9; Samulski, RJ et al. Virol. 1987; 61 (10): 3096-101). In addition, a small deletion in one of the two ITRs allows packaging of self-complementary vectors in which the genome anneals to itself after viral uncoating. This results in more efficient cell transduction, but reduces the coding capacity by half (McCarty, DM et al. Mol Ther. 2008; 16 (10): 1648-56; McCarty, DM et al. Gene Ther. 2001; 8 (16): 1248-54).

[0152] In some embodiments, a transposon / transposase system is used to deliver a polynucleotide to a cell. For example, piggyBac TM Transposon system. piggyBac TMTransposons are mobile genetic elements that efficiently transpose between vectors and chromosomes via a “cut and paste” mechanism (Woodard et al., 2015). TM The transposase recognizes the transposon-specific inverted terminal repeats (ITRs) located at both ends of the transposon vector and efficiently moves the contents from the original site and integrates them into the TTAA chromosomal locus. piggyBac TM The transposon system facilitates the efficient integration of polynucleotides into the cellular genome.

[0153] Thus, in some embodiments, the method further comprises delivering a transposon comprising the processed polynucleotide and delivering a transposase to the PSC.

[0154] In some embodiments, the processed polynucleotide is delivered into the cell using electroporation. Electroporation is a physical transfection method that uses electric pulses to create temporary holes in the cell membrane through which the processed polynucleotide can enter the cell. For example, see Chicaybam L et al. Front. Bioeng. Biotechnol., 23 January 2017.

[0155] After transfection, the processed polynucleotide can be integrated into the genome of the PSC. In some embodiments, the processed polynucleotide may further comprise an antibiotic resistance gene to confer resistance to the antibiotic used in the antibiotic drug selection process. In this way, a "pure" cell population containing the integrated processed polynucleotide can be obtained. In some embodiments, a cell population containing the integrated processed polynucleotide is selected using antibiotic drug selection. Antibiotic drug selection is the process of treating a cell population with an antibiotic so that only cells that can survive in the presence of the antibiotic will remain in the cell population. Non-limiting examples of antibiotics that can be used for antibiotic drug selection include: puromycin, blasticidin, geneticin, hygromycin, mycophenolic acid, ZEOCIN, carbenicillin, kanamycin, ampicillin and actinomycin.

[0156]

Culture medium

[0157] The methods provided herein, in some embodiments, include culturing PSCs in a feeder-free, serum-free medium. For example, the medium may include PSCs isolated from an Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., Solubilized basement membrane preparation (coated at ∼75 μl / cm 2 ~150μl / cm 2In some embodiments, the dissolved basement membrane preparation includes one or more extracellular matrix (ECM) proteins and one or more growth factors. For example, the ECM protein can be selected from laminin, collagen IV, heparan sulfate proteoglycans and entactin.

[0158] In some embodiments, the culture medium further comprises one or more growth factors, for example, selected from recombinant human basic fibroblast growth factor (rh bFGF) (e.g., 80 ng / ml to 120 ng / ml) and recombinant human transforming growth factor β (rh TGFβ) (e.g., 20 pM to 25 pM). In some embodiments, the culture medium further comprises rh bFGF and rh TGFβ. In some embodiments, the culture medium comprises mTeSR TM Plus medium (STEMCELL Technologies).

[0159] In some embodiments, the culture medium further comprises a small molecule ROCK inhibitor (eg, 9 μM to 11 μM), such as Y-27632, to facilitate seeding of PSCs.

[0160] In some embodiments, an inducing agent is added to the culture medium to produce an induction medium. In some embodiments, the induction medium comprises an inducing agent (eg, doxycycline (eg, 0.1-1 μg / mL, optionally about 0.5 μg / ml)).

[0161] [Therapeutic compositions and methods of use]

[0162] The present disclosure provides, in some embodiments, a therapeutic composition comprising microglia-like cells produced herein. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition is cryopreserved.

[0163] Such compositions can be administered to a subject, such as a human subject, using any suitable route of administration. Suitable routes of administration include, for example, parenteral routes, such as intravenous, intrathecal, parenchymal, or intraventricular routes. Suitable routes of administration include, for example, parenteral routes, such as intravenous, intrathecal, parenchymal, or intraventricular injection.

[0164] In some embodiments, the subject is a human subject.

[0165] Patients with spinal cord injury (SCI) may benefit from the use of these cells. After microglia-like cells are differentiated from iPSCs, the cells can be stimulated with IL-4 to acquire a pro-tissue repair and anti-inflammatory phenotype. The modeled microglia-like cells can then be transplanted into sites near the injury to promote neuronal repair and improve motor function.

[0166] iPSC-derived microglia can also be used as disease models to study disease pathology and screen therapeutic molecules. For example, microglia can be added to neuronal cell cultures to study the effects of immune cells on neuronal networks in response to disease stimuli (i.e., amyloid-β, tau, TDP43). Microglia can be added to three-dimensional cultures such as organoids to provide a more faithful model for brain development and degeneration. To model neurological diseases that may have a genetic background, i.e., Alzheimer's disease, ALS, or MS, microglia-like cells can be differentiated from patient-derived iPSCs to accurately reflect the patient's genetic background and allow for genetic stratification during drug screening.

[0167] These compositions can be administered to a subject in a therapeutically effective amount. The term "therapeutically effective amount" refers to the number of particles required to produce a therapeutic effect on a subject, alone or in combination with at least one other active agent. As will be appreciated by those skilled in the art, effective amounts vary, depending on the route of administration, the use of excipients, and co-use with other active agents. The dosage administered depends on the subject to be treated, including, for example, the strength of the individual's immune system or genetic susceptibility. Suitable dosage ranges are readily determined by those skilled in the art and can be in the microgram range of the polypeptides disclosed herein. The dosage of the formulations disclosed herein may depend on the route of administration and vary according to the size of the subject.

[0168] It is believed that, based on the above description, those skilled in the art can make the most of the present invention. Therefore, the following specific examples should be interpreted as being illustrative only, rather than limiting the remainder of the present disclosure in any way. All publications cited in this application are incorporated by reference for the purpose or subject matter cited in this disclosure. [Example]

[0169] Recent advances and applications of single-cell assays, exemplified by collaborative efforts such as the Human Cell Atlas (HCA), have begun to provide a comprehensive view of cell types and cell states within the human body. Such atlases are essential for understanding human development and disease. From a synthetic biology perspective, these atlases can mine promising targets for cell fate processing, with important implications for disease modeling, cell therapy, and regenerative medicine. Previously, an unbiased approach was used to screen the Comprehensive Human Transcription Factor Library (TFome) and identify 290 transcription factors (TFs) that induce differentiation of human induced pluripotent stem cells (hiPSCs) into various cell types. While unbiased screening methods have led to many interesting discoveries, it does not guarantee the generation of any specific cell type. For those who wish to differentiate stem cells into specific cell types of interest to study disease and create therapeutics, the availability of experimental and computational pipelines for identifying TFs to generate target cell types will be of great benefit. In the example below, a target cell type, microglia, for which no TF-based differentiation methods have yet been found, was selected for the development of a new screening method.

[0170] In the example below, two consecutive iterations of pooled TF screening were performed. Each round of screening consisted of the creation of a barcoded TF library, pooled transfection into iPSCs to induce differentiation, and subsequent single-cell transcriptome analysis. Based on the analysis, TFs were ranked based on their ability to induce microglial gene expression, and the top hits were then characterized based on their ability to induce differentiation into microglia ( Figure 1A , Figure 2A ). The analysis identified a TF combination that generated cells transcriptionally resembling microglia within 4 days without the need to change the culture medium. These TF-induced microglia-like cells (TFiMGL) had molecular and functional characteristics of human primary microglia. The barcoding and amplification strategy allowed the simultaneous detection of cells and TF barcodes from high-throughput single-cell experiments, enabling analysis of the regulatory relationships between TFs and other genes. A human single-cell transcriptome reference was also constructed by integrating publicly available scRNA-seq datasets from 225 samples representing 59 tissue types, onto which TF-differentiated single cells could be mapped. The methods described in this study should be widely used for cell fate engineering and enable researchers to more efficiently select TFs to generate new iPSC-derived cell types.

[0171] [Example 1: The first pooled screening identified the initial TFs that induce microglial gene expression

[0172] To identify TFs that differentiate hiPSCs into microglia, the overall strategy was to first transfect and randomly integrate a panel of TFs into cells, followed by differentiation induction and single-cell RNA sequencing (scRNA-seq). From the scRNA-seq data, TFs enriched in cells differentiating into microglia were identified based on gene expression. 40 candidate TFs were cloned into the pBAN2 vector for genomic integration with the PiggyBac transposase and doxycycline (Dox)-inducible expression. To distinguish exogenous from endogenous transcripts, a 20-nucleotide (nt) barcode was added between the stop codon and the poly A sequence of each TF. The 40 TF vectors were transfected into 600,000 hiPSCs from a healthy donor (PGP1) at a mass ratio of TF to transposase DNA of 4:1, which results in cellular uptake and integration of multiple TFs. After selection for TF-integrated cells with puromycin, differentiation was induced for 4 days ( Figure 1A Flow cytometric analysis showed that 0.3-0.5% of cells expressed microglial surface proteins, including CX3CR1, P2RY12, and CD11b ( Figure 1B ).

[0173] To determine which of the 40 TFs induced microglial gene expression, all differentiated cells were sorted for scRNA-seq ( Figure 1A After 4 days of differentiation, 30% of the cells lost the expression of the stem cell marker TRA-1-60 ( Figure 1C Two independent transfections were performed (data not shown), and approximately 10% of non-induced cells were added to each group as an undifferentiated control during scRNA-seq. Expression of microglial genes (ITGAM, P2RY12, CX3CR1, TMEM119, TREM2) was observed, as well as a group of cells that highly expressed POU5F1, a marker for stem cells ( Figure 1D , 1E ). By performing amplicon sequencing of co-amplified TFs and cellular barcodes from cDNA ( Figure 1F ), the amount of exogenous TF expressed in single cells can be quantified (data not shown). Each cell expressed an average of 7 TFs, and 877 of 10,285 single cells (8.5%) had no TF expression, which is consistent with the addition of 10% stem cells ( Figure 1G By comparing TF expression levels in cells with and without microglial RNA expression, it was possible to determine which TFs were more highly expressed in cells with microglial RNA. Three TFs were identified as potentially contributing to microglial gene expression, including SPI1, FLI1, and CEBPA ( Figure 1H). SPI1, encoding the PU.1 protein, is a known TF required for microglial development. CEBPA is a known key regulator of myeloid differentiation. FLI1, although less reported for microglial development, has been reported to interact with RUNX140 and SPI141, both of which are essential for tissue-resident macrophage development.

[0174] Expression of CEBPA and FLI1 alone in hiPSCs resulted in almost complete cell death (data not shown), indicating that their expression levels need to be tightly controlled. SPI1 alone induced only about 3% CD11b-positive cells (data not shown), indicating that additional TFs are required for differentiation. Mixed transfection of CEBPA+FLI1 (MG2.1 pool) or CEBPA+SPI1 (MG2.2 pool) resulted in improved expression of microglial markers, while CEBPA+FLI1+SPI1 (MG3.1 pool) produced the most positive cells, reaching 14% CD11b+, 54% P2RY12+ ( Fig. 1I ). However, no expression of CX3CR1, a chemokine receptor important for microglial activation and migration, was observed (43, 44). Since mixed transfection of the three plasmids and PiggyBac integration do not guarantee that each cell has all three TFs, a polycistronic expression cassette was constructed by linking the TFs to a 2A self-cleaving peptide. The position of the genes in the cassette affects their relative expression levels, with the first gene being the highest (Liu, Z. et al. Sci. Rep. 7, 2193 (2017)). TFs are arranged in different orders, and the structures are named according to the order of the TF first letters. For example, SPI1-T2A-FLI1-P2A-CEBPA was named "MG3.1-SFC". In MG3.1-CFS, -FCS, and -SFC, the first two constructs caused dramatic cell death on day 4 (data not shown), an observation consistent with the fact that CEBPA and FLI1 themselves cause cell death. MG3.1-SFC positioned SPI1 anteriorly, yielding 37% CD11b+ and 6% P2RY12+ cells, while CX3CR1 was still not expressed ( Fig. 1I ). The differences between MG3.1-SFC and MG3.1-pool may be due to different doses of TFs. While MG3.1-pool allows for variable dose combinations of the three TFs between different cells, MG3.1-SFC produces a fixed dose for all cells. Regardless of dose variation, the lack of CX3CR1 expression and the overall low percentage of CD11b and P2RY12-positive cells suggest that additional TFs are required to drive microglia differentiation from hiPSCs.

[0175] [Example 2: The second iteration identified additional TFs for microglial differentiation

[0176] To build on the hits from the first pooled screen and identify more TFs that can promote microglial differentiation, a second pooled screen was performed on the top three TFs (3+X) from the first iteration ( Figure 2A ). The second TF library contained a total of 42 candidate TFs. To ensure that each cell received both SPI1-T2A-FLI1-P2A-CEBPA and other TFs from the second library, bleomycin was used in addition to puromycin to select for both constructs ( Figure 2B ). Similar to the first pooled screen, 600,000 PGP1 hiPSCs were transfected and drug selected in duplicate. After 4 days of Dox-induced differentiation, the cell pools were subjected to single-cell RNA and TF barcode amplicon sequencing identical to the first iteration, but without cell sorting. As a control, 5% undifferentiated hiPSCs and 5% MG3.1-SFCs were added during the single-cell encapsulation process to mark the starting point of differentiation for both iterations. TF barcode analysis indeed revealed two cell clusters corresponding to hiPSCs and MG3.1-SFCs on UMAP, while also showing new cell clusters expressing additional TFs ( Figure 2C , 2D ), indicating that the differentiation of new TFs has changed. Based on TF barcode counts, among 8051 single cells from two independent transfections, 549 (6.8%) cells had no TF barcodes and 613 (7.6%) cells had only MG3.1-SFC barcodes. On average, each cell expressed 5 TFs ( Figure 2E , 2F), where the majority of cells expressed the SFC box and at least one other TF.

[0177] To determine if any novel TFs were improving microglial differentiation, we analyzed their effects on microglial RNA expression, especially CX3CR1, which is not expressed in MG3.1-SFCs. A significant increase in the number of MEF2C and KLF6 barcodes was observed in cells expressing CX3CR1 ( Figure 2G ), indicating that they are able to induce CX3CR1 expression. MEF2C was also present in the first screen, but did not reach significance in upregulating CX3CR1, suggesting that the presence of the SFC triple TF cassette is helpful. MEF2C also reached a high ranking in TMEM119 ( Figure 2G ), but lower than BHLHE41. The SFC cassette ranked highest in inducing ITGAM and P2RY12 expression, as expected from the results of the first iteration. CEBPB and IRF8 also showed the potential to promote ITGAM or P2RY12 expression ( Figure 2G). In summary, other TFs of interest include MEF2C, CEBPB, IRF8, KLF6, and BHLHE41.

[0178] To verify that additional TFs could promote microglial gene expression, they were expressed alone in SFC (SFC+1). Compared with MG3.1-SFC, CEBPB increased the percentage of CD11b+ cells from 37% to 98% ( Figure 2H ), but resulted in more cell death on day 4. MEF2C and IRF8 increased P2RY12 expression from 6% to around 45% ( Figure 2H Most importantly, MEF2C and KLF6 increased CX3CR1+ cells from zero to 20% and 2% ( Figure 2H ). These results were in good agreement with the predictions from single-cell TF barcoding analysis, demonstrating the validity of using pooled TF screens to infer causal relationships between TFs and target gene expression. To test whether microglial differentiation could be further promoted by delivering more TFs per cell, we combined MEF2C, CEBPB, and IRF8 into polycistronic cassettes. Since MEF2C showed the ability to induce CX3CR1 and P2RY12, it was placed first, and the positions of CEBPB and IRF8 were varied, resulting in two cassettes: MIC and MCI ( Fig.2I The positions of FLI1 and CEBPA were also changed in the first constructs to generate SFC and SCF, keeping SPI1 in the front to avoid excessive cell death during differentiation. All four combinations of the three-TF cassette (SFC-MIC, SFC-MCI, SCF-MIC, SCF-MCI) were tested for their ability to induce microglial differentiation ( Fig.2I ). Encouragingly, all six-TF mixtures generated cell pools with increased expression of microglial proteins compared to MG3.1-SFC ( Figure 2J ). The best mixture was MG6.4-SCF-MCI, containing 66% CD11b+, 93% P2RY12+, and 16% CX3CR1+ cells on day 4, while for MG3.1-SFC these numbers were 37% / 6% / 0%. These results highlight the importance of the second iteration and demonstrate the utility of iterative TF screens in cell fate engineering.

[0179] [Example 3: TFiMGLs differentiate rapidly, are phagocytic, respond to disease-related stimuli, and share molecular features with primary microglia]

[0180] To determine the differentiation kinetics of MG6.4, bulk RNA-seq analysis was performed on cells at 0, 1, 2, 3, 4, and 6 days after induction. Rapid induction of the six TFs was observed on day 1, reaching a plateau on day 2 ( Figure 3A This was accompanied by a rapid downregulation of POU5F1 on day 1, followed by upregulation of microglial genes starting on day 2 ( Figure 3B ). Principal component analysis (PCA) reflected a similar trend, with rapid differentiation occurring on days 1 and 2, followed by a gradual slowing down from days 3 to 6 ( Figure 3C ). The transcriptomes at day 4 and day 6 were closely linked, indicating a stable window for functional studies and applications. Therefore, for downstream characterization of MG6.4, all differentiations were performed for 4 days.

[0181] Brightfield microscopy analysis of MG6.4 revealed rapid morphological changes from day 1 to day 6 (data not shown). Immunofluorescence analysis confirmed the expression of key TFs and microglial proteins ( Figure 3D MG6.4 showed reproducible differentiation between replicates, with 53.9±0.57% CD11b+, 93.1±0.50% P2RY12+, and 14.8±0.68% CX3CR1+ cells ( Figure 3E ). As brain-resident macrophages, microglia play an important role in brain development and homeostasis. The ability of microglia to respond to signals associated with degenerating neurons and phagocytes is integral to their function. To investigate whether MG6.4 could mimic the phagocytic function of microglia, MG6.4 was incubated with pHrodo Green-labeled Staphylococcus aureus (S. aureus) particles for 0.5, 2, and 4 hours, followed by flow cytometry and microscopy analysis. While the 0.5 hour incubation showed minimal phagocytic activity, almost all cells were positive for pHrodo Green at 2 hours, and the intensity became stronger at 4 hours ( Figure 3F Microscopic analysis of co-staining for microglial surface proteins at 4 h confirmed the intracellular location of these particles ( Figure 3G To investigate whether MG6.4 responds to ADP stimulation, on day 4 MG6.4 were pre-incubated with the calcium indicator Fluo-4 and stimulated with medium containing ADP while imaging every three seconds. A rapid increase in calcium signal was observed upon addition of ADP ( Figure 3H , 3I , 3J), indicating that MG6.4 responds to ADP stimulation.

[0182] To evaluate how TFiMGL recapitulates the transcriptome of human microglia, previously published large amounts of RNA-seq data for human primary microglia and iPSC-derived microglia were downloaded (Abud, EM et al. Neuron 94, 278-293.e9 (2017); Gosselin, D. et al. Science 356, (2017); Galatro, TF et al. Nat. Neurosci. 20, 1162-1171 (2017)). To address potential batch issues that may hinder meaningful comparisons between datasets, all original FASTQ files were aligned to the same reference genome, and the batch effect correction method ComBat-seq50 based on negative binomial regression was applied before downstream analysis. According to PCA analysis, MG6.4 on days 2 to 6 were closer to primary microglia from different sources than iPSCs or monocytes, indicating that microglial fate induction was successful ( Figure 4A ). scRNA-seq data from primary human brains can define gene sets that are enriched in microglia. Using two microglia gene sets in MSigDB derived from human brain scRNA-seq, MG6.4 vs. iPSC gene set enrichment analysis (GSEA) was performed on day 4 (Subramanian, A. et al. Proc. Natl. Acad. Sci. USA 102, 15545-15550 (2005)). Positive microglia gene enrichment scores were observed using both sets, indicating that MG6.4 upregulated those microglia-enriched genes compared to iPSCs ( Figure 4B ). A set of 881 microglia-enriched genes (Gosselin, D. et al. Science 356, (2017)) was used to Figure 4A Although MG6.4 on day 1 still clustered closer to iPSCs, MG6.4 on days 2 and beyond clustered closer to primary microglia, with most genes being more upregulated on days 4 and 6 than on days 2 and 3 (data not shown). These results suggest that MG6.4 is able to establish a microglia-like transcriptome starting from day 2.

[0183] Microglia are able to respond to signals indicative of brain infection and inflammation. IFNγ is a known activator of microglia secreted by T lymphocytes. Amyloid β (Aβ), a key molecule in AD pathology, can trigger microglial responses. Aggregation of TDP-43 is considered a hallmark of ALS and is present in the vast majority of ALS patients and has also been shown to activate microglia. To investigate how TFiMGL respond to IFNγ, fibrillar Aβ (fAβ), and TDP-43, day 4 MG6.4 were treated with stimuli in triplicate for 24 h and harvested for RNA-seq. PCA analysis showed transcriptome changes in the IFNγ and TDP-43 treated groups, while the fAβ treated group had minimal differences ( Figure 4C ). The formation of fAβ was confirmed by in vitro amyloid fibrillation assay, which showed that Aβ peptides could form fibrils after 1 h of incubation (data not shown). Pathway analysis of differentially expressed genes in the IFNγ-treated group included “response to viruses” and “response to bacteria” ( Figure 4D ), which is expected because IFNγ is produced by T cells in response to infection. The genes with the highest upregulation by IFNγ included CXCL10, CXCL11, IRF1, and IL18BP (data not shown). For TDP-43-treated cells, the major differentially regulated pathways included “myeloid leukocyte-mediated immunity” and “myeloid cell activation involved in the immune response” ( Figure 4E ), further confirming the activation of MG6.4 by TDP-43. The genes with the highest upregulation by TDP-43 included CXCL1, SOD2, NFKBIZ, CCL20, and C3 (data not shown). Overall, these results suggest that MG6.4 exhibits the expected response to infection and ALS-related stimuli. The lack of response to fAβ treatment remains to be investigated.

[0184] [Example 4: Single-cell atlas reference atlas identifies causal TFs for multiple cell fates]

[0185] Determining cell types using a panel of cellular markers is a common practice in studies of primary human tissue and stem cell differentiation. Recent advances in single-cell analysis technologies have made it possible to define cell types based on more comprehensive molecular profiles, including transcriptome, epigenome, proteome, and metabolome. To investigate how single-cell atlas data can be used to guide cell fate engineering efforts, several prerequisites are currently only partially met: (1) the existence of single-cell data from all human tissue types and, ideally, also at different developmental stages; (2) data integration methods to combine datasets from different sources to create a comprehensive cell atlas; (3) reference mapping methods to map new datasets (e.g., iPSC-derived cells) onto the atlas and quantitatively score their similarity to all classes; and (4) the existence of perturbation libraries compatible with scRNA-seq, including but not limited to open reading frame (ORF) and CRISPR libraries.

[0186] To explore the idea of ​​atlas-guided cell fate engineering, scRNA-seq data from published datasets generated by the 10× Chromium platform were compiled using the two merged TF screens described previously for microglial differentiation, with the majority of data coming from PanglaoDB55, where all raw reads from different studies were aligned and processed together. Two additional organs underrepresented in PanglaoDB, namely brain and endometrium, were added. The final single-cell atlas contained a total of 225 samples from 59 organ / tissue types, for a total of 1,004,650 single cells (data not shown). All raw data downloaded from PanglaoDB were carefully filtered in Seurat for cells, genes, UMI numbers, and mitochondrial gene ratios (data not shown). In its current state, the atlas is annotated to the organ / tissue level, and obtaining cell-level annotations from all 59 studies may be a work in progress to improve the atlas. To reduce batch variations caused by different studies, the data were integrated with two different pipelines, Clustered Similarity Profiles (CSS) or Harmony (data not shown). Qualitative evaluation of the integrated UMAP plots indeed indicated co-clustering of cells in related tissues from different studies, such as a mix of the “primary brain” and “embryonic forebrain” datasets and the “islet” and “pseudoislet” datasets (data not shown). Next, scRNA-seq data from the two merged TF screens were mapped onto the integrated maps using CSS or Symphony60 (data not shown). Nearest neighbor (NN) analysis in the CSS or Harmony corrected meta-feature space revealed similarities in the most likely primary tissue types of cells driven by TFs (data not shown).

[0187] At least in the case of TF-driven iPSC differentiation described here, being identified as a NN or colocalizing on a UMAP plot does not mean that the processed cells share the exact transcriptome of the primary cells. The interpretation is that when a NN is identified, the processed cells share the most gene expression signatures with their NNs compared to all other reference cells on a relative scale with current sensitivity and accuracy. Despite the removal of cells with the stem cell marker TRA-1-60+ prior to scRNA-seq during the first pooled screen, the majority of cells were mapped to the “iPSC / NPC” sample, indicating that these cells were incompletely differentiated (data not shown). This proportion continued to decrease in the second pooled screen when analyzed by computational pipelines (data not shown), which was expected because most cells had already integrated with the first three hits (SPI1, FLI1, CEBPA) from the first iteration. The driving TFs that caused the processed cells to localize on organs / tissues other than “iPSC / NPC” were then investigated. From both pipelines, a group of cells mapped to “primary brain”. Given that microglia are the most abundant myeloid cells in the brain, it was hypothesized that these cells might be targeted to microglia. Obtaining cell-level annotations for the “primary brain” dataset showed that these processed cells were closely connected to cells annotated as “microglia” in the study (data not shown). Analysis of TF barcodes with high expression in the “microglia” mapping group showed that CEBPA and CEBPB were significantly enriched (data not shown), partially confirming the hits identified from both screening iterations. CEBPA was also enriched in cells mapped to the “bone marrow whole”, “PBMC”, and “monocyte-derived macrophage” samples (data not shown). CIITA was highly enriched in the “cord blood CD34” sample (data not shown). Other NN-enriched TFs were also analyzed (data not shown). Based on the exploratory experiments and analyses presented here, it is reasonable to expect that with larger perturbation libraries, more refined single-cell maps, and dedicated computational pipelines, it will be possible to explore cell fate processing in a more multiplexed manner.

[0188]

method

[0189]

Construction of TF expression vector with barcode

[0190] All TFs used in this study were obtained from the TFome collection in pDONR format. For expression in hiPSCs, PiggyBac integrating the Dox-inducible vector pBAN was used. To prepare the barcoded pBAN expression vector (pBAN-BC), the original pBAN was digested with AgeI and KpnI, and then gBlock (IDT DNA) containing the same excised fragment and an additional 20 bp random barcode was ligated. After bacterial transformation, individual colonies were amplified and plasmid DNA was extracted. Each TF was transferred from pDONR to the pBAN-BC vector using Gateway cloning. The barcode sequence of each TF was confirmed by Sanger sequencing.

[0191]

Cell culture

[0192] hiPSCs were cultured in mTeSR Plus medium (Stemcell Technologies, 100-0276) on multi-well plates coated with Matrigel (Corning, 354277) or Cultrex (Bio-Techne Corporation, 3434-005-02). For passaging, cells were dissociated with TrypLE Express (Life Technologies, 12604013) and inoculated into fresh plates and medium containing 10 μM Y-27632ROCK inhibitor (Millipore, 688001) for 24 hours. The medium was changed every day until the cells were ready for another passaging or downstream experiments.

[0193]

Nucleofection, TF integration and differentiation

[0194] TF (pBAN-TF-BC) and Super PiggyBac (SPB) transposase (System Biosciences, PB210PA-1) expression vectors were mixed at a 4:1 mass ratio and transfected into hiPSCs using the P3 Primary Cell 4D-Nucleofector X Kit L (Lonza, V4XP-3024) on a 4D-Nucleofector X unit (Lonza, AAF-1002X) according to the manufacturer's instructions. For two pooled TF screens, 600,000 cells were transfected with 5 μg DNA and seeded into one well of a 6-well plate. For a single TF combination, 120,000 cells were transfected with 2.5 μg DNA and seeded into one well of a 12-well plate. Program CB150 was used for nucleofection. 48 hours after nuclear transfection, 1 μg / mL puromycin (Gibco, A1113803) or 50 μg / mL ZEOCIN (Gibco, R25001) was added to the culture to screen for TF integrated cells. When 80% confluence was reached, the cells were passaged again. To induce TF expression, cells were inoculated into mTeSR Plus medium containing 0.5 μg / mL doxycycline (Sigma-Aldrich, D3072) and 10 μM Y-27632 ROCK inhibitor, and replaced with medium containing only doxycycline after 24 hours.

[0195] Flow cytometry and sorting

[0196] For cell analysis, cells were dissociated with TrypLE Express at 37°C for 5 minutes, diluted with two volumes of cell staining buffer (Biolegend, 420201), and centrifuged at 200g for 3 minutes to remove digestive enzymes. The cells were then incubated on ice for 15 minutes with 25 μg / mL human Fc Block (BD Biosciences, 564219) diluted with cell staining buffer and immediately stained on ice for 30 minutes with appropriately diluted fluorescent-conjugated antibodies or isotype controls. Antibodies were diluted in cell staining buffer, and human Fc Block was not removed from the mixture. After antibody staining, cells were washed twice with cell staining buffer and then placed in 5 mL round-bottom polystyrene tubes (Falcon, 352235) through a 35 μm nylon mesh. Flow cytometry data were collected on a BD LSRFortessa cell analyzer. For cell sorting, the staining protocol was the same, except that the cell staining buffer was replaced with mTeSR Plus medium to maintain optimal cell viability. Cell sorting was performed on a BD FACSAria cell sorter. Flow cytometry antibodies used in this study were: FITC-TRA-1-60 (BD Biosciences, 560380), BV421-CX3CR1 (Biolegend, 341620), PE-P2RY12 (Biolegend, 392104), APC-CD11b (Biolegend, 101212). Isotype controls used were: BV421-rat IgG2b (Biolegend, 400640), PE-mouse IgG2a (Biolegend, 400214), APC-rat IgG2b (Biolegend, 400612).

[0197]

scRNA-seq library preparation

[0198] scRNA-seq experiments were performed using 10× Genomics Chromium Single Cell 3′ Reagent Kits v3 or v3.1 following the manufacturer’s instructions. 5000 single cells were calculated as the target starting amount for each sample. In the first iteration, 10% stem cells were added as an undifferentiated control. In the second iteration, 5% stem cells and 5% MG3.1-SFC were added as undifferentiated and initial differentiation controls. The only modification made to the protocol was that during the sample index PCR step, 5 μL of the PCR mix was removed and mixed with 0.5 μL of 1000× SYBR Gold (Invitrogen, S11494) for the qPCR reaction. The optimal amplification cycle was determined as the cycle before the half-peak of the total signal. The final library was sequenced on a NextSeq 500 or NovaSeq, with a goal of at least 30,000 reads per cell.

[0199]

TF barcode amplicon library preparation

[0200] Because the amplicons contain cellular barcodes, UMIs, and TF barcodes after the cDNA amplification step in the 10× scRNA-seq protocol, these cDNAs can be used as templates for further amplification of TF cellular barcodes. Two consecutive PCR reactions were performed, each followed by qPCR with the addition of SYBR Gold to determine the optimal number of cycles, as described in “scRNA-seq library preparation”. For PCR1, NGS10x-F-i7-BC-PCR1F and i5000 were used as primers. A 50 μL PCR1 reaction volume contained 25 μL Q5 Hot Start High Fidelity 2× Master Mix (New England Biolabs, M0494L), 5 μL amplified cDNA, 2.5 μL of two primers (10 μM stock concentration), and 15 μL nuclease-free water. The PCR1 program was initial denaturation at 98°C for 30 seconds; 11 to 13 cycles of 98°C for 10 seconds, 67°C for 30 seconds, and 72°C for 30 seconds (qPCR assay); and a final extension at 72°C for 2 minutes. The PCR1 reaction was purified using 1.2X SPRIselect beads (Beckman Coulter, B23318) according to the standard protocol. The sample was eluted with 20 μL of water. For PCR2, i7000, P5, and P7 were used as primers. A 50 μL PCR2 reaction contained 25 μL Q5 Hot Start High Fidelity 2× Master Mix, 10 μL PCR1 product, 2.5 μL all three primers (10 μM stock concentration), and 7.5 μL nuclease-free water. The PCR2 program was initial denaturation at 98°C for 30 seconds; 4-5 cycles of 98°C for 10 seconds, 67°C for 30 seconds, and 72°C for 30 seconds (qPCR assay); and a final extension at 72°C for 2 minutes. The PCR2 product was purified in the same manner as PCR1. The final library was submitted to MiSeq v3, and the paired ends were read for 80 cycles in either direction.

[0201]

Primer sequence

[0202]

[0203]

Analysis of scRNA-seq and TF barcode-seq data

[0204] For scRNA-seq, raw FASTQ files were aligned to GRCh38 and quantified using Cell Ranger. Seurat was used to perform cell filtering, data normalization, and clustering. The resulting Seurat object also contained the single-cell raw expression matrix for all genes. For TF barcode sequences, in paired-end MiSeq data, one read pair contained a 20bp TF barcode, while the other read pair contained a 16bp cell barcode and a 12bp UMI. The three sequences were compiled into a table with three columns by matching the names of the reads in the pair: TF-BC, cell-BC, UMI. To remove duplicate reads from the same molecule, duplicate rows with the same values ​​for all three columns were removed. The table was then counted and reshaped into a frequency table, where the row names represent cells and the column names represent TFs. This table contains the raw counts of each TF barcode in all individual cells. Since TF barcodes are amplified from cDNA during library preparation, we normalized the TF barcode counts using the number of total RNA UMIs detected in each cell, reasoning that cells with more total UMIs are likely to have more TF barcode reads. The raw gene expression matrix and the normalized TF count matrix were used to identify which TF barcodes may induce microglial gene expression. Specifically, the expression of microglial genes was binarized, with any cell with non-zero expression being 1. A Wilcoxon rank sum test was then performed on all barcode TFs between the two groups of cells with 0 or 1 microglial gene expression to determine which TFs were more highly expressed in cells expressing microglial genes. TFs were ranked by -log10 (p-value).

[0205]

Batch RNA-seq library preparation

[0206] Cultured cells were directly dissolved with TRIzol (Thermo Fisher Scientific, 15596018) for purification of total RNA using the Direct-zol RNA MiniPrep Kit (Zymo Research, R2050). RNA concentration was quantified using the Qubit RNAHS Assay Kit (Thermo Fisher Scientific, Q32852). RNA integrity was confirmed by the presence of 18S and 28S bands on a 2% E-Gel EX agarose gel (Thermo Fisher Scientific, G402002). 100 ng to 1000 ng of total RNA was used as the starting amount for mRNA enrichment using the NEBNext Poly (A) mRNA Magnetic Separation Module (New England Biolabs, E7490), and libraries were then constructed using the NEBNext Ultra II Directed RNA Library Preparation Kit (New England Biolabs, E7760S) according to the manufacturer's instructions. Library QC and sequencing were performed at the Biopolymers Facility at Harvard Medical School.

[0207]

Large-scale RNA-seq data analysis

[0208] For samples generated in-house and datasets downloaded from GEO, raw FASTQ files were aligned to GRCh38 and quantified using STAR 2.5.2b. Regularized logarithmic (rlog) transformation was applied to raw counts before visualization using PCA. For analyses involving data from multiple sources, batch correction was performed using ComBat-seq before PCA. Differential gene expression analysis was performed using DESeq2 46 Pathway enrichment and GSEA analysis using clusterProfiler 73 .

[0209]

Immunofluorescence (IF)

[0210] IF experiments were performed in μ plate 96-well black plates (ibidi, 89626). After removing the culture medium, the cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, 15710) in 1× phosphate buffered saline (PBS) (Thermo Fisher Scientific, 10010072) at room temperature (RT) for 15 minutes. Before permeabilization or blocking, the cells were rinsed 3 times with PBS. For staining of Oct-3 / 4 and PU.1, the cells were permeabilized, while cell surface proteins were not stained. Permeabilization was performed with 0.25% Triton-X-100 (Thermo Fisher Scientific, 85111) at RT for 15 minutes in 1× PBS, followed by rinsing 3 times with PBS. The cells were then blocked with 1% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. For primary and secondary antibody staining, the antibodies were diluted in PBS containing 1% BSA and incubated with the cells for 1 hour at room temperature. After staining, the cells were washed three times for 5 minutes with PBS to remove excess antibodies. Cells were imaged directly on a Nikon Ti2 Eclipse inverted microscope equipped with a Plan ApoλDM 60× (1.4NA, Ph3) oil objective and an Andor Zyla sCMOS camera. Images were acquired using NIS-Element AR software. All antibodies were used at a 1:200 dilution. The primary IF antibodies used in this study were: Oct-3 / 4 (Santa Cruz Biotechnology, sc-5279), PU.1 (Thermo Fisher Scientific, PA5-17505), CD11b (BioLegend, 101202), P2RY12 (Thermo Fisher Scientific, 702516), CX3CR1 (Abcam, ab8021).

[0211]

Phagocytosis test

[0212] Differentiated cells were incubated with 20 μg / mL pHrodo Green Staphylococcus aureus (S. aureus) BioParticles (Thermo Fisher Scientific, P35382) in mTeSR Plus medium in the presence of 100 μg / ml penicillin-streptomycin (Corning, 30-002-CI) for 0 to 4 hours. After washing with PBS to remove excess particles, cells were harvested for antibody (CX3CR1, P2RY12, CD11b) staining and flow cytometric analysis as described in the previous section. The remaining stained cells after flow cytometry were transferred to μPlate 96-well Black plates for fluorescence microscopy to confirm the intracellular localization of the particles. This step needs to be performed quickly after flow cytometry to avoid changes in cell morphology due to cell death.

[0213]

Calcium imaging

[0214] Calcium imaging experiments were performed in standard 12-well cell culture plates. Differentiated cells were incubated with 1 μg / mL Fluo-4 AM calcium indicator (Thermo Fisher Scientific, F23917) in 1 mL mTeSR Plus medium in a cell culture incubator for 30 min. Excess dye was washed away with two 1 mL filler washes. After adding 1 mL of fresh mTeSR Plus, the cells were placed on the microscope stage in the incubator. Image acquisition began for 90 seconds without stimulation to determine the baseline signal. An image was acquired every 3 seconds, which is the fastest speed on the instrument. After 90 seconds, 1 mL of medium containing 150 μM ADP was added to the cells while imaging continued. The total imaging time was 10 minutes. The fluorescence signal was quantified and plotted in MATLAB.

[0215] [Amyloid fibrillation]

[0216] Aβ fibrillation experiments were performed using the SensoLyte Thioflavin Tβ-amyloid (1-42) Aggregation Kit (AnaSpec, AS-72214) according to the manufacturer's instructions. Reactions were set up in μ-plate 96-well Black plates. Data were collected on a microplate reader, excitation / emission = 440nm / 484nm, 37°C, every 5 minutes for 3 hours. [Preparation of data sets for construction of single-cell reference atlas]

[0217] Files containing raw counts of 10× Genomics Chromium scRNA-seq data from different human tissues were downloaded from PanglaoDB (https: / / panglaodb.se / index.html). Human primary brain single cell data from gestational weeks 6 to 22 were downloaded from Organoid Report Card (https: / / cells.ucsc.edu / ?ds=organoidreportcard). Human endometrial single cell data were downloaded from GEO GSE111976. All samples were manually cell filtered using Seurat using different filters for gene number, UMI, and mitochondrial gene percentage. Tissue annotations were compiled by manually curating each study by checking which tissues / cell types were used. All raw count tables were merged into a sparse matrix and then used as input for data integration.

[0218] Single-cell atlas integration and mapping

[0219] Data integration and mapping using CSS or Harmony / Symphony were performed following the authors' instructions on GitHub (https: / / github.com / quadbiolab / simspec; https: / / github.com / immunogenomics / harmony; https: / / github.com / immunogenomics / symphony). Due to the size of the data, these steps were performed on the O2 cluster at Harvard Medical School, which has at least 180Gb of memory and 8 cores. Most R objects in the pipeline can be saved as standard R files for repeated analysis, with the exception of UMAP model files, which need to be saved and loaded via the "uwot" package. The code used for integration and mapping, as well as key reference and annotation files that can be used for future exploration, are shared with this manuscript.

[0220] All references, patents, and patent applications disclosed herein are incorporated by reference to the subject matter to which they are addressed, which in some cases may be the entire document.

[0221] The indefinite articles "a" and "an" used in the specification and claims should be understood to mean "at least one" unless expressly stated otherwise.

[0222] It should also be understood that, unless explicitly stated to the contrary, in any method claimed in this document, comprising multiple steps or acts, the order in which the steps or acts of the method are recited is not necessarily limited to the order in which the steps or acts of the method are recited.

[0223] In the claims and the foregoing specification, all transitional phrases, such as "comprises," "including," "carrying," "having," "containing," "involving," "holding," "consisting of," and the like, shall be construed as open ended, i.e., meaning but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the U.S. Patent Office Manual of Patent Examining Procedures.

[0224] The terms "about" and "substantially" preceding a numerical value mean ±10% of the recited numerical value.

[0225] Where a range of values ​​is provided, every value between the upper and lower limits of the range, inclusive, is specifically contemplated and described herein.

Claims

1. A pluripotent stem cell (PSC), comprising: one or more processed polynucleotides, wherein the processed polynucleotides comprise open reading frames encoding SPI1 protein, FLI1 protein and CEBPA protein.

2. The PSC according to claim 1, wherein The one or more processed polynucleotides comprise a first polycistronic polynucleotide comprising: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the FLI1 protein, and a third open reading frame encoding the CEBPA protein.

3. The PSC according to claim 1, wherein The one or more processed polynucleotides comprise a first polycistronic polynucleotide, which comprises the following open reading frames, ordered from 5' to 3' direction of the first polycistronic polynucleotide: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the FLI1 protein, and a third open reading frame encoding the CEBPA protein.

4. The PSC according to claim 1, wherein The one or more processed polynucleotides comprise a first polycistronic polynucleotide, which comprises the following open reading frames, arranged in a 5' to 3' direction of the first polycistronic polynucleotide: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the CEBPA protein, and a third open reading frame encoding the FLI1 protein.

5. A PSC according to any one of the preceding claims, wherein: The one or more processed polynucleotides comprise an open reading frame encoding one or more of a MEF2C protein, a CEBPB protein and an IRF8 protein.

6. The PSC according to claim 5, wherein The one or more processed polynucleotides comprise open reading frames encoding two or more of the MEF2C protein, the CEBPB protein and the IRF8 protein.

7. The PSC according to claim 6, wherein The one or more processed polynucleotides comprise open reading frames encoding MEF2C protein, CEBPB protein and IRF8 protein.

8. The PSC according to claim 7, wherein The one or more processed polynucleotides comprise a second polycistronic polynucleotide, which comprises the following open reading frames in the 5' to 3' direction of the second polycistronic polynucleotide: a first open reading frame encoding the MEF2C protein, a second open reading frame encoding the CEBPB protein and a third open reading frame encoding the IRF8 protein.

9. The PSC according to claim 7, wherein The one or more processed polynucleotides comprise a second polycistronic polynucleotide, which comprises the following open reading frames in the 5' to 3' direction of the second polycistronic polynucleotide: a first open reading frame encoding the MEF2C protein, a second open reading frame encoding the IRF8 protein and a third open reading frame encoding the CEBPB protein.

10. The PSC of claim 1, wherein: The one or more processed polynucleotides comprise: A first polycistronic polynucleotide, which optionally comprises from 5′ to 3′: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the CEBPA protein, and a third open reading frame encoding the FLI1 protein; and The second polycistronic polynucleotide optionally comprises from 5′ to 3′: a first open reading frame encoding the MEF2C protein, a second open reading frame encoding the CEBPB protein, and a third open reading frame encoding the IRF8 protein.

11. A PSC according to any one of the preceding claims, wherein: The one or more open reading frames of the one or more processed polynucleotides are operably linked to a heterologous promoter.

12. The PSC according to claim 11, wherein The heterologous promoter is an inducible promoter.

13. The PSC according to claim 12, wherein The inducible promoter is a chemically inducible promoter.

14. A pluripotent stem cell (PSC), comprising: SPI1 protein, FLI1 protein and CEBPA protein, wherein the proteins are overexpressed.

15. The PSC of claim 14, wherein: The PSC further comprises one or more of a MEF2C protein, a CEBPB protein, and an IRF8 protein, wherein one or more of the MEF2C protein, the CEBPB protein, and the IRF8 protein are overexpressed.

16. The PSC of claim 15, wherein: The PSC further comprises two or more of a MEF2C protein, a CEBPB protein, and an IRF8 protein, wherein two or more of the MEF2C protein, the CEBPB protein, and the IRF8 protein are overexpressed.

17. The PSC of claim 15, wherein: The PSC further comprises a MEF2C protein, a CEBPB protein and an IRF8 protein, wherein the MEF2C protein, the CEBPB protein and the IRF8 protein are overexpressed.

18. A PSC according to any one of the preceding claims, wherein: The PSCs are human PSCs.

19. A PSC according to any one of the preceding claims, wherein: The PSCs are induced PSCs (iPSCs).

20. A composition comprising a population of PSCs according to any one of the preceding claims.

21. A method for producing a microglia-like cell population, comprising: culturing a population of pluripotent stem cells (PSCs) in a culture medium to produce an expanded population of PSCs; and SPI1 protein, CEBPA protein and FLI1 protein are expressed in PSCs of the expanded population.

22. The method according to claim 21, wherein: The PSCs of the amplified population contain more than one processed polynucleotide, wherein the processed polynucleotide contains open reading frames encoding SPI1 protein, FLI1 protein and CEBPA protein.

23. The method according to claim 22, wherein: The one or more processed polynucleotides comprise a first polycistronic polynucleotide comprising: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the FLI1 protein, and a third open reading frame encoding the CEBPA protein.

24. The method according to claim 22, wherein: The one or more processed polynucleotides comprise a first polycistronic polynucleotide, which comprises the following open reading frames, ordered from 5' to 3' direction of the first polycistronic polynucleotide: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the FLI1 protein, and a third open reading frame encoding the CEBPA protein.

25. The method according to claim 22, wherein: The one or more processed polynucleotides comprise a first polycistronic polynucleotide, which comprises the following open reading frames, arranged in a 5' to 3' direction of the first polycistronic polynucleotide: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the CEBPA protein, and a third open reading frame encoding the FLI1 protein.

26. The method according to any one of the above claims, further comprising expressing one or more of MEF2C protein, CEBPB protein and IRF8 protein in the PSCs of the expanded population.

27. The method according to any one of claims 22 to 26, wherein: The one or more processed polynucleotides also comprise an open reading frame encoding one or more of a MEF2C protein, a CEBPB protein, and an IRF8 protein.

28. The method according to any one of the above claims, further comprising expressing two or more of the MEF2C protein, the CEBPB protein and the IRF8 protein in the PSCs of the expanded population.

29. The method according to any one of claims 22 to 28, wherein: The one or more processed polynucleotides also comprise open reading frames encoding two or more of the MEF2C protein, the CEBPB protein and the IRF8 protein.

30. The method of any one of the above claims, further comprising expressing MEF2C protein, CEBPB protein and IRF8 protein in the PSCs of the expanded population.

31. The method according to any one of claims 22 to 29, wherein: The one or more processed polynucleotides also comprise open reading frames encoding MEF2C protein, CEBPB protein and IRF8 protein.

32. The method according to any one of claims 22 to 29, wherein: The one or more processed polynucleotides also comprise a second polycistronic polynucleotide, which further comprises the following open reading frames, arranged in the 5' to 3' direction of the second polycistronic polynucleotide: a first open reading frame encoding the MEF2C protein, a second open reading frame encoding the CEBPB protein, and a third open reading frame encoding the IRF8 protein.

33. The method according to any one of claims 22 to 29, wherein: The one or more processed polynucleotides also comprise a second polycistronic polynucleotide, which further comprises the following open reading frames, arranged in the 5' to 3' direction of the second polycistronic polynucleotide: a first open reading frame encoding the MEF2C protein, a second open reading frame encoding the IRF8 protein, and a third open reading frame encoding the CEBPB protein.

34. The method of claim 21, wherein: The PSCs of the expanded population comprise: A first polycistronic polynucleotide, which optionally comprises from 5′ to 3′: a first open reading frame encoding the SPI1 protein, a second open reading frame encoding the CEBPA protein, and a third open reading frame encoding the FLI1 protein; and The second polycistronic polynucleotide optionally comprises from 5′ to 3′: a first open reading frame encoding the MEF2C protein, a second open reading frame encoding the CEBPB protein, and a third open reading frame encoding the IRF8 protein.

35. A method according to any one of the preceding claims, wherein: The one or more open reading frames of the one or more processed polynucleotides are operably linked to a heterologous promoter.

36. The method of claim 35, wherein: The heterologous promoter is an inducible promoter.

37. A method according to any one of the preceding claims, wherein: The PSC population contains 1×10 2 ~1×10 7 PSCs.

38. A method according to any one of the preceding claims, wherein: The PSC population is cultured for about 2 to 5 days.

39. The method of claim 38, wherein: The PSC population was cultured for approximately 4 days.

40. A method according to any one of the preceding claims, wherein: Microglia-like cells of the microglia-like cell population are CD11b + 、CX3CR1 + 、ITGAM + 、P2RY12 + 、TMEM119 + and / or TREM2 + .

41. A method according to any one of the preceding claims, wherein: TRA-1-60 - and / or POU5F1 - .

42. Producing CD11b + 、CX3CR1 + 、ITGAM + 、P2RY12 + 、TMEM119 + TREM2 + TRA-1-60 - and / or POU5F1 - A method for producing microglia-like cells, comprising: (a) delivering one or more processed polynucleotides to pluripotent stem cells (PSCs), the processed polynucleotides comprising an inducible promoter operably linked to one or more open reading frames encoding SPI1 protein, CEBPA protein and FLI1 protein, optionally wherein the one or more processed polynucleotides further comprise an inducible promoter operably linked to one or more open reading frames encoding MEF2C protein, CEBPB protein and / or IRF8 protein; and (b) culturing the expanded population of PSCs in an induction medium comprising an induction agent.

43. Producing CD11b + 、CX3CR1 + 、ITGAM + 、P2RY12 + 、TMEM119 + TREM2 + TRA-1-60 - and / or POU5F1 - A method for producing microglia-like cells, comprising: (a) delivering one or more processed polynucleotides to pluripotent stem cells (PSCs), the processed polynucleotides comprising an inducible promoter operably linked to one or more open reading frames encoding SPI1 protein, CEBPA protein and FLI1 protein, optionally wherein the one or more processed polynucleotides further comprise an inducible promoter operably linked to one or more open reading frames encoding MEF2C protein, CEBPB protein and / or IRF8 protein; (b) seeding the PSCs in a feeder-free, serum-free culture medium, and optionally culturing the PSCs for about 1 to about 24 hours; and (c) The PSCs of (b) are cultured in an induction medium containing an induction agent.

44. The method of claim 42 or 43, comprising delivering to the PSC: (i) a first polycistronic polynucleotide comprising a first inducible promoter operably linked to an open reading frame encoding SPI1, an open reading frame encoding CEBPA, and an open reading frame encoding FLI1, and (ii) a second polycistronic polynucleotide comprising a second inducible promoter operably linked to an open reading frame encoding MEF2C, an open reading frame encoding CEBPB and an open reading frame encoding IRF8.

45. The method according to any one of claims 42 to 44, wherein: The first and / or second polycistronic polynucleotide is a transposon, and the delivering further comprises delivering a transposase to the PSC.

46. ​​The method according to any one of claims 42 to 45, wherein: The first and / or second inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

47. The method according to any one of claims 42 to 46, wherein: (b) Feeder-free, serum-free medium contains a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

48. The method of claim 47, wherein: The solubilized basement membrane preparation includes extracellular matrix (ECM) proteins and growth factors.

49. The method of claim 48, wherein: The ECM protein is selected from the group consisting of laminin, collagen IV, heparan sulfate proteoglycans, and entactin.

50. The method according to any one of claims 42 to 49, wherein: The feeder-free, serum-free culture medium further comprises a growth factor selected from the group consisting of recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ).

51. The method according to any one of claims 42 to 50, wherein: (c) The expanded population of PSCs is approximately 20,000 cells / cm 2 ~60,000 cells / cm 2 Density culture.

52. The method according to any one of claims 43 to 51, wherein: The feeder-free, serum-free medium of (b) also contains a small molecule ROCK inhibitor.

53. The method according to any one of claims 42 to 52, wherein: Such inducers include doxycycline.

54. The method according to any one of claims 42 to 53, wherein: The PSCs are cultured in the induction medium for about 72 to about 96 hours.

55. Microglia-like cells produced by the method of any one of the preceding claims.

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