Reagents and methods for producing erythromyeloid progenitors, NK cells and megakaryocytes

Abstract

The present disclosure provides reagents and methods for producing NK progenitor cells, NK cells, megakaryocytes, and their CAR derivatives from hematopoietic stem cells. Pharmaceutical compositions containing the NK progenitor cells produced by the reagents and methods of the present invention, and immunotherapy using these pharmaceutical compositions are also provided.

Description

[Technical Field]

[0001] Federally Supported Research This invention was made with government support under awards HL142665, OD011106 and HL134655 given by the National Institutes of Health. The government has certain rights in this invention. Sequence Listing Description A computer-readable format of the Sequence Listing has been submitted with this application by electronic filing and is incorporated by reference in its entirety. The Sequence Listing is contained in a file with the file name "22-0629-WO.xml" created on September 7, 2023, and is 37kb in size. [Background technology]

[0002] Over the past 25 years, elucidation of developmental pathways using human embryonic stem cells and induced pluripotent stem cells has enabled, among other things, the production of therapeutically useful quantities of specific cell types that can provide a homogeneous source of specific cells for individual patients. Specific examples of such cell types include those produced during hematopoiesis to provide many important components of blood function and homeostasis, such as cells involved in immunological surveillance and function. One such cell is the natural killer cell, or NK cell, a type of lymphoid lineage white blood cell characterized by possessing granules containing enzymes that can kill tumor cells or virus-infected cells. Yet another is the megakaryocyte, a cell found in the bone marrow involved in producing platelets. Another important aspect of our ability to study and manipulate stem cells, and hematopoietic stem cells specifically, is understanding the endogenous proteins produced during differentiation that influence their progression to final effector cells, such as NK cells. These proteins, including transcription factors, are involved in activating specific genes in developmental pathways that lead to specific differentiation outcomes. One example of such a transcription factor is the SOXF family of transcription factors, including SOX7, SOX17, and SOX18, which are recognized as key regulators of angiogenesis, cardiovascular development, and hematopoietic development. See, e.g., Lilly et al., 2017 SOXF transcription factors in cardiovascular development. Seminars in cell & developmental biology 63: 50-57.

[0003] Studies in mouse embryos have shown that Sox7 is required for the formation of early multipotent hematopoietic progenitors (HPs) with erythromyeloid differentiation potential. Gandillet et al., 2009, Sox7-sustained expression alters the balance between proliferation and differentiation of hematopoietic progenitors at the onset of blood specification. Blood. 114: 4813-4822. Forced expression of Sox7 in cells derived from E7.5 mouse embryos or in vitro-differentiated mouse embryonic stem cells (ESCs) results in early CD41 expression with erythromyeloid differentiation potential. +It promotes the self-renewal of hematopoietic progenitors and blocks their differentiation. Lilly et al., 2016, Interplay between SOX7 and RUNX1 regulates hemogenic endothelial fate in the yolk sac. Development 143: 4341-4351. A similar phenotype was observed after overexpression of Sox18 in in vitro differentiated mouse ESCs. Serrano et al., 2010, Contrasting effects of Sox17- and Sox18-sustained expression at the onset of blood specification. Blood 115: 3895-3898. Using human embryonic stem cells, SOX17 was shown to be a key regulator of HOXA and the arterial program in hemogenic endothelial cells (HE), resulting in robust lymphoid-myeloid differentiation potential and DLL4 expression, similar to arterial HE, at the site of HSC emergence. + CXCR4 + It is required for the specification of phenotypic HE. Jung et al., 2021, SOX17 integrates HOXA and arterial programs in hemogenic endothelium to drive definitive lympho-myeloid hematopoiesis. Cell Rep. 34(7):108758. There is a need in the art to further elucidate the function of transcription factors in the SOXF family to develop reagents and methods for manipulating hematopoiesis in human stem cells to produce cells for therapeutic intervention and other purposes. Summary of the Invention

[0004] Provided herein are reagents and methods for manipulating pluripotent stem cells, particularly human stem cells, to produce specific cell types resulting from hematopoietic differentiation. In certain embodiments, such cells are natural killer (NK) cells. In other specific embodiments, they are megakaryocytes. Certain embodiments of the methods provided herein result in forced expression of SOX18 in stem cells and hemogenic endothelial cells during the endothelial-to-hematopoietic transition. In particular, such forced expression of SOX18 results in preferential commitment to NK progenitor cells. In certain embodiments, such NK progenitor cells express CD34 + CD43 + CD235a / CD41a - CD45 - In a specific embodiment, the present invention provides such cells for use, inter alia, in immunotherapy. Yet another aspect of the present disclosure provides a pharmaceutical composition comprising NK cells produced by forced expression of SOX18 in hemogenic endothelial cells. In some embodiments, the NK cells are genetically modified cells, such as CAR-NK cells. In certain embodiments, an expression construct encoding a CAR is introduced into pluripotent stem cells (PSCs) and the cells differentiated into CAR-NK cells by forced expression of SOX18. In alternative embodiments, NK cells are produced from PSCs, and an expression construct encoding a CAR is introduced into the NK cells thereby produced.

[0005] In yet a further aspect of the present disclosure, megakaryocytes are provided through forced expression of SOX18 in mesodermal and hemogenic endothelial cells. These and other features, objects, and advantages of the present invention will be better understood from the ensuing description. In this description, reference is made to the accompanying figures, which form a part of the description and which illustrate by way of example, and not limitation, embodiments of the present invention. The description of preferred embodiments is not intended to limit the invention, which covers all modifications, equivalents, and alternatives. Accordingly, reference should be made to the claims recited herein to interpret the scope of the invention. The present disclosure will be better understood, and features, aspects and advantages in addition to those described above will become apparent, when considered in light of the following detailed description, which refers to the following drawings: [Brief explanation of the drawings]

[0006] [Figure 1] FIG. 1 is a diagram of the vector used in the PiggyBac system to generate DOX-inducible SOX18 in the H1 hESC line. [Figure 2A] Figure 2 shows the effect of forced expression of SOX-18 on hematopoietic differentiation of human pluripotent stem cells (hPSCs). Figure 2A shows a timeline of hematopoietic differentiation, where D represents the day of differentiation. The cell types resulting after treatment of the developmental cell cultures listed horizontally are indicated at each developmental day below the timeline. [Figure 2B] Figure 2B shows the effect of forced expression of SOX-18 on hematopoietic differentiation of human pluripotent stem cells (hPSCs). Figure 2B shows the experimental design. Cells were differentiated after the indicated DOX treatments. Floating HPs were collected on differentiation day 8 and evaluated for phenotype, CFC, and lymphoid differentiation potential. [Figure 2C] Figure 2C shows the effect of forced expression of SOX-18 on hematopoietic differentiation of human pluripotent stem cells (hPSCs). Figure 2C shows flow cytometry count plots illustrating the phenotype of hematopoietic progenitor cells isolated from D8 differentiation cultures of DOX-induced SOX18 hPSCs. [Figure 2D] Figure 2 shows the effect of forced expression of SOX-18 on hematopoietic differentiation of human pluripotent stem cells (hPSCs). Figure 2D illustrates the percentage of CD34+CD43+ cells and the composition of CD34+CD43+ subsets at D8 of differentiation (results are mean ± SD, n=5 for two independent experiments performed in triplicate and duplicate). *p<0.05, **p<0.01, and ***p<0.001, one-way ANOVA, Tukey's multiple comparison test). [Figure 2E]Figure 2B shows the effect of forced expression of SOX-18 on hematopoietic differentiation of human pluripotent stem cells (hPSCs). Figure 2C shows the CFC differentiation potential of D8 HPs. Results are mean ± SD, n = 2. **p < 0.01, two-way ANOVA. [Figure 2F] Figure 2F shows the effect of forced expression of SOX-18 on hematopoietic differentiation of human pluripotent stem cells (hPSCs). Figure 2F shows flow cytometry analysis of DOX-induced T cell differentiation in differentiated human pluripotent stem cells (hPSCs). [Figure 2G] Figure 2G shows the effect of forced expression of SOX-18 on hematopoietic differentiation of human pluripotent stem cells (hPSCs). The total number of T cells generated from 104 D8 CD43+ cells and the percentage of CD5+CD7+ and CD4+CD8+ cells are shown (results are mean ± SD, n = 5 experiments). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, one-way ANOVA, Dunnett's multiple comparison test). [Figure 2H] Figure 2H shows the effect of forced expression of SOX-18 on hematopoietic differentiation of human pluripotent stem cells (hPSCs). Figure 2H shows flow cytometry analysis of NK cell differentiation induced by DOX in differentiating hPSCs. [Figure 2I] Figure 2I shows the effect of forced expression of SOX-18 on hematopoietic differentiation of human pluripotent stem cells (hPSCs). Figure 2I is a graphical representation showing the total number of CD56+ cells generated from 104 CD43+ cells and the expression of CD94 and CD16 by NK cells (results are mean ± SD, n = 6 for two independent experiments performed in triplicate). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA, Dunnett's multiple comparison test. [Figure 3A] Figures 3A-3E show experimental results demonstrating that SOX18 has a limited effect on the specification of hemogenic endothelial cells. Figure 3A shows flow cytometry results demonstrating the expression of arterial markers and Venus in VEC+SOX18 cells at day 4 of differentiation with or without DOX. [Figure 3B]Figures 3A-3E show experimental results demonstrating that SOX18 has a limited effect on hemogenic endothelial cell specification. Figures 3B and 3C are graphical representations showing the percentage and total number of VEC+, DLL4+CXCR4+, and VEC+DLL4+CXCR4- cells generated from 10,000 hPSCs on day 4 of differentiation. Results are shown as mean ± SD, n = 3. ***p < 0.001, t-test. [Figure 3C] Figures 3A-3E show experimental results demonstrating that SOX18 has a limited effect on hemogenic endothelial cell specification. Figures 3B and 3C are graphical representations showing the percentage and total number of VEC+, DLL4+CXCR4+, and VEC+DLL4+CXCR4- cells generated from 10,000 hPSCs on day 4 of differentiation. Results are shown as mean ± SD, n = 3. ***p < 0.001, t-test. [Figure 3D] Figures 3A-3E show experimental results demonstrating that SOX18 has a limited effect on the specification of hemogenic endothelial cells, and Figure 3D shows flow cytometry results demonstrating the expression of arterial markers and Venus in differentiated D4 VEC+iSOX18 cells with or without DOX. [Figure 3E] Figures 3A-3E show experimental results demonstrating that SOX18 has a limited effect on the specification of hemogenic endothelial cells. Figures 3E and 3F are graphical representations showing the percentage and total number of VEC+, VEC+DLL4+CXCR4+, and VEC+DLL4+CXCR4- cells generated from 104 hPSCs at differentiation day 5 with or without DOX (results shown as mean ± SD, n = 3). ***p<0.001, t-test; **p<0.01, two-way ANOVA, Sidak's multiple comparison test). [Figure 3F]Figures 3A-3E show experimental results demonstrating that SOX18 has a limited effect on the specification of hemogenic endothelial cells. Figures 3E and 3F are graphical representations showing the percentage and total number of VEC+, VEC+DLL4+CXCR4+, and VEC+DLL4+CXCR4- cells generated from 104 hPSCs at differentiation day 5 with or without DOX (results shown as mean ± SD, n = 3). ***p<0.001, t-test; **p<0.01, two-way ANOVA, Sidak's multiple comparison test). [Figure 4A] Figure 4A shows the results of an experiment illustrating that SOX18 overexpression affects lymphoid specification from HE cells by shifting the balance between NK and T lymphocyte differentiation. Figure 4A is a schematic diagram illustrating the experimental design described in Example 3. [Figure 4B] Figure 4B shows the results of an experiment illustrating that SOX18 overexpression affects lymphoid specification from HE cells by shifting the balance between NK and T lymphocyte differentiation. Figure 4B is a graphical representation of the CFC differentiation potential of HP generated from D4 HE after 5 days of co-culture with OP9 or DLL4-OP9 with or without DOX. Results are expressed as mean ± SD, n = 2. **p < 0.01 and ****p < 0.0001, two-way ANOVA, Tukey's multiple comparison test. [Figure 4C] Figure 4A shows the results of an experiment illustrating that SOX18 overexpression affects lymphoid specification from HE cells by shifting the balance between NK and T lymphocyte differentiation. Figure 4C shows a graphical representation of the total number of CD4+CD8+ T cells generated from 10,000 CD43+ cells. Results are expressed as mean ± SD, n = 4. ****p < 0.0001, two-way ANOVA, Sidak's multiple comparison test. [Figure 4D] Figure 4D shows the results of an experiment illustrating that SOX18 overexpression affects lymphoid specification from HE cells by shifting the balance between NK and T lymphocyte differentiation. Figure 4D shows flow cytometry results demonstrating the effect of SOX18 overexpression on T cell differentiation from hemogenic endothelial cells. [Figure 4E] Figure 4B shows the results of an experiment illustrating that SOX18 overexpression affects lymphoid specification from HE cells by shifting the balance between NK and T lymphocyte differentiation. Figure 4E is a graphical representation of the total number of CD56+ NK cells generated from 10,000 CD43+ cells. Results are expressed as mean ± SD, n = 4. ****p < 0.0001, two-way ANOVA, Sidak's multiple comparison test. [Figure 4F] Figure 4F shows the results of an experiment illustrating that SOX18 overexpression affects lymphoid specification from HE cells by shifting the balance between NK and T lymphocyte differentiation. Figure 4F shows flow cytometry results demonstrating the effect of SOX18 overexpression on NK cell differentiation from hemogenic endothelial cells. [Figure 5A] 5A-5G illustrate the characterization of NK cell differentiation potential in iSOX18 hPSCs. Figure 5A is a diagram of the experimental design showing the DOX treatment schedule and cell subset analysis described in Example C. [Figure 5B] Figures 5A-5G illustrate the characterization of NK cell differentiation potential in iSOX18 hPSCs. Figure 5B shows flow cytometry results demonstrating the NK cell differentiation potential of cell subsets isolated from cultures in the presence and absence of DOX. [Figure 5C] Figures 5A-5G illustrate the characterization of NK cell differentiation potential in iSOX18 hPSCs. Figure 5C is a graphical representation of the total number of CD56+ NK cells generated from 10,000 cells from the indicated cell subsets. Results are expressed as mean ± SD, n = 4. ****p<0.0001, one-way ANOVA, Tukey's multiple comparison test. [Figure 5D] Figures 5A-5G illustrate the characterization of NK cell differentiation potential in iSOX18 hPSCs. Figure 5D is a graphical representation of the total number of CD56+ cells in cultures from DOX-free and DOX2-8 HP after 1 and 4 weeks of differentiation in NK cultures (results are mean ± SD, n = 10-11). ****p<0.0001, two-way ANOVA, Sidak's multiple comparison test. [Figure 5E] Figures 5A-5G illustrate the characterization of NK cell differentiation potential in iSOX18 hPSCs. Figure 5E is a graphical representation of the proliferation curves of CD56+ cells in NK differentiation cultures initiated from D8 CD43+ cells generated in DOX-free and DOX2-8-treated cultures. Cell cultures were initiated with 105 CD43+ cells and continued for 5 weeks (results are mean ± SD, n = 2). [Figure 5F] Figures 5A-5G illustrate the characterization of NK cell differentiation potential in iSOX18 hPSCs. Figure 5F is a graphical representation of a cytotoxicity assay against K562 targets. Varying amounts of target cells (effector:target ratios, 1:1 to 5:1) were plated with CD56+ cells for 4 hours (results are mean ± SD, n = 2 experiments). [Figure 5G] Figures 5A-5G illustrate the characterization of NK cell differentiation potential in iSOX18 hPSCs. Figure 5G is a flow cytometry count plot showing CD107a and interferon gamma (IFNγ) expression by CD56+ cells after K562 stimulation. [Figure 5H] Figures 5A-5G illustrate the characterization of NK cell differentiation potential in iSOX18 hPSCs. Figure 5H is a graphical representation of the percentage of CD107a+IFNγ- or CD107a-IFNγ+ cells after PMA / ionomycin stimulation. Results are expressed as mean ± SD. *p<0.05, t-test. [Figure 6A] Figures 6A-6E illustrate molecular profiling of HE, HP, and NK cells generated from iSOX18 hPSCs. Figure 6A shows flow cytometry results illustrating cell populations isolated for RNA-seq analysis. [Figure 6B] Figures 6A-6E illustrate molecular profiling of HE, HP, and NK cells generated from iSOX18 hPSCs. Figure 6B is a graphical representation showing the number of DEGs in the indicated cell populations. [Figure 6C]Figures 6A-6E illustrate molecular profiling of HE, HP, and NK cells generated from iSOX18 hPSCs. Figure 6C is a heatmap showing differentially expressed genes in D4 HE P1 populations from cultures with and without DOX. [Figure 6D] Figures 6A-6E illustrate molecular profiling of HE, HP, and NK cells generated from iSOX18 hPSCs. Figure 6D shows a bar plot illustrating the presence of significantly enriched KEGG pathways in selected differential expression comparisons. The y-axis indicates the enriched pathway category, and the x-axis indicates the number of significantly enriched genes in each pathway. [Figure 6E] Figures 6A-6E illustrate molecular profiling of HE, HP, and NK cells generated from iSOX18 hPSCs. Figure 6E is a bar graph showing KEGG pathways significantly enriched in selected differential expression comparisons. The y-axis indicates the enriched pathway category, and the x-axis indicates the number of significantly enriched genes in each pathway. [Figure 6F] Figures 6A-6E illustrate molecular profiling of HE, HP, and NK cells generated from iSOX18 hPSCs. Figure 6F shows the fold change of selected genes in two KEGG pathways that were significantly enriched in D8 P1 DOX+ cells compared with D8 P1 DOX-less cells. [Figure 7A] Figure 7 illustrates DOX-induced SOX18 production in the H1 hESC line: Figure 7A shows flow cytometry analysis of undifferentiated iSOX18 cells cultured with or without DOX. [Figure 7B] Figure 7 illustrates DOX-induced SOX18 production in the H1 hESC line. Figure 7B is a fluorescent image showing the expression of the Venus reporter in undifferentiated iSOX18 cells cultured with or without DOX. The scale bar is 200 μM. [Figure 7C]Figure 7 illustrates DOX-induced SOX18 production in the H1 hESC line. Figure 7C shows qRT-PCR analysis demonstrating SOX18 expression in D4 HE generated in iSOX18 hPSC differentiation cultures with or without DOX. Results are mean ± SD (N=9). ****p<0.0001, t-test. [Figure 7D] Figure 7 illustrates DOX-induced SOX18 production in the H1 hESC line. Figure 7D is a Western blot showing upregulation of SOX18 expression in undifferentiated iSOX18 cells 24 hours after DOX treatment and in D5 differentiated iSOX18 hPSCs treated with DOX from D2 to D5. [Figure 8A] Figures 8A and 8B provide the results of a detailed analysis of the D8 CD43+ subset recovered from cultures with or without DOX using a t-distributed stochastic neighbor embedding algorithm (t-SNE). Figure 8A shows an overlay of t-SNE plots of the DOX-free and DOX2-8 CD43+ subsets at D8. Each dot represents one cell. [Figure 8B] Figure 8A provides the results of a detailed analysis of the D8 CD43+ subset recovered from cultures with or without D2-8 DOX using a t-distributed stochastic neighbor embedding algorithm (t-SNE). Figure 8B shows that the no-DOX or DOX2-8 samples produced a single tSNE plot using a single surface marker. Marker expression levels are indicated using a continuous grayscale scale. [Figure 8C] Figure 8A provides the results of a detailed analysis of the D8 CD43+ subset recovered from cultures with or without D2-8 DOX using a t-distributed stochastic neighbor embedding algorithm (t-SNE). Figure 8C shows tSNE maps generated for the no-DOX and DOX2-8 subsets. Each shade represents a D8 CD43+ no-DOX or DOX2-8 cell. Each dot represents a cell, and cells are shaded according to their assigned subset. Cell populations are defined by a manual gating strategy. [Figures 9A-9C] Figure 9 shows the effect of SOX18 overexpression on megakaryocyte and hemoheoangioblast (HB) development. Figure 9A shows count plots demonstrating megakaryocytic marker expression in megakaryocytic differentiation cultures initiated using CD43+ cells recovered from DOX-free and DOX2-8 cultures at D8. Figure 9B is a graphical representation of the percentage of megakaryocytic cells generated in these cultures. Results are shown as mean ± SD, n = 3. **p < 0.01 and ***p < 0.001, t-test. Figure 9C is a graphical representation of the HB-CFC differentiation potential of iSOX18 cells. Cells were recovered from D3 differentiation with no DOX or D2-3 DOX treatment. The graph shows the number of HB colonies per 10,000 cells recovered at D3 of differentiation. Results are shown as mean ± SD, n = 2. **p < 0.01, t-test. [Figure 10A] Figure 10 illustrates the effect of SOX18 overexpression on cell cycle and apoptosis. Figure 10A shows the analysis of gene enrichment in the GO "apoptosis". [Figure 10B] Figure 10 illustrates the effect of SOX18 overexpression on cell cycle and apoptosis. Figure 10B shows an analysis of gene enrichment in the GO "control of cell population proliferation" gene set in the indicated cell subsets in DOX-treated and untreated cultures. Shaded areas indicate significant enrichment in DOX-treated versus untreated cultures. [Figure 10C] Figure 10 illustrates the effect of SOX18 overexpression on cell cycle and apoptosis. Figure 10C shows representative dot plots of flow cytometry analysis of the cell cycle of D5 HE with DOX and DOX-free iSOX18 hPSC cultures. [Figure 10D] Figure 10 illustrates the effect of SOX18 overexpression on cell cycle and apoptosis. Figure 10D is a bar graph showing the mean +-SD (D) of duplicate experiments for flow cytometry analysis of the cell cycle of D5 HE in DOX and DOX-free iSOX18 hPSC cultures. ***p<0.001, and ****p<0.0001, one-way ANOVA Dunnett's multiple comparison test. DETAILED DESCRIPTION OF THE INVENTION

[0007] The present disclosure is based, at least in part, on experimental demonstration that forced expression of SOX18 in cells of the hematopoietic lineage preferentially promotes the formation of natural killer cell and megakaryocyte progenitor cells. For the purposes of promoting an understanding of the principles of the present disclosure, reference will be made herein to embodiments, and specific language will be used to describe the principles of the present disclosure. Nevertheless, no limitation of the scope of the present disclosure is intended thereby, and it will be understood that variations and further modifications of the present disclosure as exemplified and contemplated herein will normally occur to those skilled in the art to which the present disclosure pertains.

[0008] definition As used herein, the articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means at least one element and may include more than one element. "About" is used to provide flexibility to the endpoints of a numerical range by allowing a given value to be "slightly above" or "slightly below" the endpoint without affecting the desired result. The term "about" in reference to a numerical value means that the numerical value may vary by no more than plus or minus 5% of the numerical value. Throughout this specification, unless the context requires otherwise, the words "comprise" and "include" and variations (e.g., "comprises," "comprising," "includes," "including") will be understood to imply the inclusion of a stated component, property, element or step or group of components, properties, elements or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0009] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or"). Unless otherwise indicated herein, the recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each discrete value within the range. Furthermore, each discrete value is incorporated into the specification as if it were individually set forth herein. For example, if a range is stated as 1 to 50, this is intended to indicate that values ​​such as 2 to 4, 10 to 30, or 1 to 3, etc., are specifically recited in the present disclosure. These are merely examples of what is specifically intended, and all possible combinations between the numerical values, including the lowest and highest values ​​recited, are considered to be specifically recited in the present disclosure. The term "contacting" includes physical contact of at least one substance with another substance.

[0010] As used herein, "treatment" refers to a clinical intervention performed in response to a disease, disorder, or physiological condition in a subject, or to which the subject is susceptible. The goals of treatment include alleviating or preventing symptoms, slowing or halting the progression or worsening of the disease, disorder, or condition, and / or ameliorating the disease, disorder, or condition. The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce a beneficial or desired biological and / or clinical result. In other words, a "therapeutically effective" amount is an amount that results in some relief, alleviation, or reduction of at least one clinical symptom in a subject. The terms "express" or "expression" refer to the transcription and translation of a nucleic acid coding sequence resulting in the production of the encoded polypeptide. "Express" or "expression" also refers to an antigen being expressed on the surface of a cell. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0011] As used herein, the term "subject" refers to both human and non-human animals. The term "non-human animal" of the present disclosure includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Human subjects can be of any age (e.g., infants, children, or adults). The term "construct" refers to an artificially designed segment of DNA that can be used to incorporate genetic material into target cells (e.g., hPSCs). The term "sequence identity" refers to the number of identical or similar nucleotide bases in a comparison between a test and a reference oligonucleotide or nucleotide sequence. Sequence identity can be determined by sequence alignment of a first nucleic acid sequence to a second nucleic acid sequence to identify similar or identical regions. As described herein, sequence identity is generally determined by alignment to identify identical residues. Matches, mismatches, and gaps can be identified between compared sequences by techniques well known in the art. Alternatively, sequence identity can be determined without considering gaps as the number of identical positions divided by the length of all aligned sequences x 100. In one embodiment, the term "at least 90% sequence identity" refers to a percent identity of 90-100% compared to a reference nucleotide sequence. For illustrative purposes, identity at a level of 90% or greater refers to the fact that, assuming a comparison of test and reference polynucleotide sequences 100 nucleotides in length, 10% or less (i.e., 10 out of 100) of the nucleotides in the test oligonucleotide differ from those in the reference oligonucleotide. Differences are defined as nucleic acid substitutions, insertions or deletions.

[0012] Any suitable method can be used to detect the expression of biological markers characteristic of the cell types described herein. For example, the presence or absence of one or more biological markers can be detected using, for example, RNA sequencing (e.g., RNA-seq), immunohistochemistry, polymerase chain reaction, quantitative real-time PCR (qRT-PCR), or other techniques for detecting or measuring gene expression. RNA-seq is a high-throughput sequencing technology that provides a genome-wide assessment of the RNA content of an organism, tissue, or cell. Alternatively or additionally, the presence or absence of, or the level of, one or more biological markers of HPC can be detected or measured using, for example, fluorescent in situ hybridization (FISH; see WO98 / 45479, published October 1998), Southern blot, Northern blot, or polymerase chain reaction (PCR) techniques, such as qRT-PCR. In exemplary embodiments, cell populations obtained by the methods provided herein are assessed for the expression (or absence) of biological markers of HPCs, such as CD34, CD45, CD43, and CD90. Quantitative methods for assessing marker expression at the protein level in cell populations are also well known in the art.

[0013] As used herein, the term "genetically engineered" refers to cells that have been manipulated using biotechnology to alter the genetic makeup of the cell, including gene transfer within and across species boundaries to produce improved or non-naturally occurring cells. Human pluripotent stem cells, hematopoietic endothelial cells, megakaryocytes, or NK cells that contain exogenous, recombinant, synthetic, and / or other modified polynucleotides are considered to be genetically engineered and therefore non-naturally occurring cells compared to any naturally occurring counterpart. In some cases, genetically engineered cells contain one or more recombinant nucleic acids. In other cases, genetically engineered cells contain one or more synthetic or genetically engineered nucleic acids (e.g., a nucleic acid that contains at least one artificially created insertion, deletion, inversion, or substitution compared to the sequence found in its naturally occurring counterpart). Procedures for producing genetically engineered cells are generally well known in the art and are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual (Fourth Edition), Cold Spring Harbor Press, Cold Spring Harbor, NY (2012) and Doudna et al., CRISPR-Cas, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2016).

[0014] Genetically engineered cells can be cells that have been modified using gene editing technology. Gene editing refers to a type of genetic manipulation in which DNA is inserted, deleted, modified, or replaced in the genome of a living cell. In contrast to other genetic engineering techniques that may randomly insert genetic material into the host genome, gene editing can target insertion to a site-specific location (for example, AAVS1 allele). Examples of gene editing techniques include, but are not limited to, restriction enzymes, zinc finger nucleases, TALEN, and CRISPR-Cas9. Genetically engineered cells can be either stem cells (e.g., human pluripotent stem cells) or their differentiated progeny (e.g., mesodermal cells, hemangioblasts, hemogenic endothelial cells, hematopoietic progenitor cells, megakaryocytes, and NK cells) that have been modified to express. Any cell described herein can be genetically engineered. In some embodiments, genetically engineered cells refer to cells differentiated from genetically engineered cells. As used herein, the term "tumor cell" refers to an abnormal cell that is constantly dividing. In some embodiments, a tumor cell is a solid tumor cell. A solid tumor is an abnormal mass of cells that typically does not contain cysts or liquid areas. Examples of solid tumors include, but are not limited to, sarcomas and carcinomas. Blood cancers (e.g., leukemia) do not typically form solid tumors. In some embodiments, a "tumor cell" is not a blood cancer cell. As used herein, a "tumor cell" refers to a group of tumor cells and / or a single tumor cell.

[0015] Human pluripotent stem cells (hPSCs), either embryonic or artificial, provide access to early stages of human development and offer a platform for deriving large numbers of hematopoietic progenitor or blood cells for cell therapy and tissue engineering. Accordingly, methods provided herein can include differentiating human pluripotent stem cells under conditions that promote differentiation of mesodermal cells (e.g., arterial endothelial cells) into hematopoietic progenitor cells, megakaryocytes, and NK cells. Methods for differentiating hPSCs into mesodermal progenitor cells, hemogenic endothelial cells, and hematopoietic progenitor cells are well known in the art. In exemplary embodiments, the culture medium used in any of the differentiation methods described above comprises IF9S medium described herein. In one embodiment, the IF9S medium used is IF9S medium comprising the formulation set forth in Table 2. In some embodiments, any of the above-described reference cells (e.g., human pluripotent stem cells) are cultured on tenascin-C. In some embodiments, any reference cell is cultured at a density of 10,000 cells / cm of the substrate. 2In some embodiments, the tenascin-C used is human tenascin-C. In some embodiments, the substrate is at least about 0.25 μg / cm 2 ~1μg / cm 2 , e.g., 0.4 μg / cm 2 , 0.5 μg / cm 2 , 0.7 μg / cm 2 , 0.8 μg / cm 2 or at least about 0.25 μg / cm 2 ~1μg / cm 2 In a preferred embodiment, the cells are cultured on plates coated with collagen IV as described in Uenishi et al., 2014, Stem Cell Reports 3: 1073-1084 and U.S. Pat. No. 9,938,499.

[0016] In some embodiments, the concentrations in the cell culture medium used in the differentiation methods described herein are: BMP4 is about 50 ng / ml to about 250 mg / ml; Activin A is about 10 ng / ml to about 15 ng / ml; FGF2 is about 10 ng / ml to about 50 ng / ml; LiCl is about 1 mM to about 2 mM; VEGF is about 20 ng / ml to about 50 ng / ml; SCF is about 50 ng / ml to about 100 ng / ml; TPO is about 50 ng / ml to about 100 ng / ml; IL-6 is about 50 ng / ml to about 100 ng / ml; and IL-3 is about 5 ng / ml to about 15 ng / ml. In some embodiments, any of the above-referenced cells are cultured in a xeno-free cell culture medium. Of critical importance for clinical treatment is the absence of xenogeneic materials in the derived cell population, i.e., the absence of non-human cells, cell fragments, serum, proteins, etc. Preferably, the present invention leads to xeno-free differentiated cells through the use of tenascin-C or collagen IV as a platform, essentially replacing the contact with OP9 cells used in early differentiation systems. In addition, the media disclosed herein are synthetic and, in some embodiments, made xeno-free, incorporating human proteins that can be produced using recombinant technology or derived from placenta or other human tissues in place of animal-derived proteins. In some embodiments, all proteins added to the medium are recombinant proteins.

[0017] As used herein, the term "mesodermal cells" refers to cells that have mesoderm-specific gene expression and can differentiate into mesodermal lineages, such as bone, muscle, including cardiac muscle, skeletal muscle, and smooth muscle (e.g., of the intestine), connective tissue, such as dermis and cartilage, kidney, urogenital system, blood or hematopoietic cells, heart, and vasculature. Mesoderm-specific biomarkers include brachyury (T). Culturing can be performed on any suitable surface (e.g., two-dimensional or three-dimensional culture). The medium and substrate conditions for culturing pluripotent stem cells used in the methods described herein are well known in the art. In some cases, pluripotent stem cells differentiated by the methods disclosed herein are cultured in mTESR-1 medium (StemCell Technologies, Inc., Vancouver, British Columbia), E8 medium, or Essential 8 medium (Life Technologies, Inc.) on MATRIGEL™ substrate (BD Biosciences, NJ) or vitronectin (Life Technologies) according to the manufacturer's protocol.

[0018] As used herein, the term "albumin-free conditions" indicates that the culture medium used does not contain any form of added albumin, including, but not limited to, bovine serum albumin (BSA), any form of recombinant albumin, or any other animal albumin. As used herein, the terms "synthetic medium" and "synthetic culture medium" also refer to a culture medium containing a fully disclosed or identifiable formula of components, the exact amounts of which are known or identifiable and can be individually adjusted. Thus, a culture medium is not synthetic if (1) the chemical and structural identity of all medium components is not known, (2) it contains any component in unknown amounts, or (3) both. Standardizing culture conditions by using a synthetic culture medium minimizes the possibility of lot-to-lot or batch-to-batch variation in the materials to which cells are exposed during cell culture. Thus, the effects of various differentiation factors become more predictable when added to cells and tissues cultured under synthetic conditions. As used herein, the term "serum-free" refers to cell culture materials that do not contain serum or serum substitutes, or that are essentially free of serum or serum substitutes. For example, an essentially serum-free medium may contain less than about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% serum. "Serum-free" also refers to culture components that do not contain serum obtained from animal (e.g., fetal bovine) blood or animal-derived materials, which is important for reducing or eliminating the possibility of cross-species viral or prion transmission. For the avoidance of doubt, serum-containing medium is not chemically defined.

[0019] Human pluripotent stem cells (e.g., human ESCs or iPS cells) can be cultured in the absence of a feeder layer (e.g., a fibroblast feeder layer) in a conditioned medium or a culture medium containing poorly defined or unknown components. As used herein, "feeder-free" refers to a culture condition that is substantially free of a cell feeder layer. Cells grown under feeder-free conditions can grow on a substrate, e.g., a synthetic substrate, and / or can grow as adherent cultures. Suitable synthetic substrates include vitronectin. A method for producing hematopoietic progenitor cells can include culturing human pluripotent stem cells in a serum-free, albumin-free, defined culture medium that promotes differentiation into mesoderm, wherein the pluripotent stem cell-derived mesodermal cells are differentiated according to the HPC differentiation methods provided herein, thereby producing pluripotent stem cell-derived HPCs.

[0020] As used herein, "pluripotent stem cells" suitable for use in the methods of the present invention are cells that have the ability to differentiate into cells of all three germ layers. Pluripotent cells suitable for use herein include human embryonic stem cells (hESCs) and human induced pluripotent stem (iPS) cells. As used herein, "embryonic stem cell" or "ESC" refers to a single pluripotent cell or a population of pluripotent cells derived from the inner cell mass of a blastocyst. See Thomson et al., Science 282:1145-1147 (1998). These cells can express Oct-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Pluripotent stem cells appear as compact colonies containing cells with a high nucleus-to-cytoplasm ratio and prominent nucleoli. ESCs are commercially available from sources such as WiCell Research Institute (Madison, WI).

[0021] As used herein, "induced pluripotent stem cells" or "iPS cells" refer to pluripotent cells or populations of pluripotent cells that differ from the differentiated somatic cells of their origin, differ in a particular set of potency-determining factors, and differ in the culture conditions used to isolate them, but are substantially genetically identical to the respective differentiated somatic cells of their origin, and yet exhibit characteristics similar to higher potency cells, such as ESCs, as described herein. See, e.g., Yu et al., Science 318:1917-1920 (2007). Induced pluripotent stem cells exhibit similar morphological characteristics (e.g., round shape, large nucleoli, and scant cytoplasm) and growth characteristics (e.g., doubling time of approximately 17-18 hours) to ESCs. In addition, iPS cells express markers specific to pluripotent cells (e.g., Oct-4, SSEA-3, SSEA-4, Tra-1-60, or Tra-1-81, but not SSEA-1). However, induced pluripotent stem cells are not directly derived from an embryo. As used herein, "not directly derived from an embryo" means that the starting cell type for producing iPS cells is a non-pluripotent cell, e.g., a multipotent cell or a highly differentiated cell, e.g., a somatic cell obtained from a postnatal individual.

[0022] As described herein, NK cells and megakaryocytes can be preferentially produced relative to T cells by inducing expression of SOX18 in progenitor cells. In general, preferential differentiation can be achieved by overexpressing SOX18 at a specific time during differentiation (i.e., starting on day 2) and for a defined period of time (i.e., 2-8 days). SOX18 is a member of the Sry-related high-mobility group (SOX) family of transcription factors and is a key developmental regulator of endothelial and hematopoietic lineages. In the methods of the present invention, differentiating hPSCs are forced to overexpress SOX18 during mesodermal differentiation by introducing an inducible SOX18 transgene into the progenitor cell population. The SOX18 transgene used in the present invention can include any nucleic acid sequence encoding the SOX18 protein. For example, the SOX18 transgene can be obtained by amplifying the SOX18 gene sequence from a genomic locus in human cells, or by amplifying SOX18 mRNA from hPSCs differentiated into endothelial cells and blood cells and converting it into cDNA. Alternatively, genomic DNA or cDNA clones can be obtained commercially (e.g., from Sino Biological, Origene, or IDT). In some embodiments, the transgene includes SEQ ID NO: 16, a cDNA sequence encoding human SOX18 protein (having the amino acid sequence set forth in SEQ ID NO: 17).

[0023] In some embodiments, the SOX18 transgene further comprises a vector sequence that can be used to drive expression of the SOX18 transgene in cells. In these embodiments, the transgene is introduced into a population of hPSCs by transducing the cells with the vector. As used herein, the term "vector" refers to a nucleic acid molecule that can propagate another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors." A vector suitable for use in the present invention contains a nucleotide sequence encoding a SOX18 transgene and heterologous sequences necessary for normal propagation of the vector and expression of the encoded polypeptide. The heterologous sequences (i.e., sequences derived from a species different from that of the transgene) may include a heterologous promoter or a heterologous transcriptional control region that allows expression of the polypeptide. Vectors suitable for expression of the SOX18 transgene include plasmids and viral vectors. In a preferred embodiment, the vector contains heterologous sequences that allow transient and / or inducible expression of the encoded SOX18 protein.

[0024] In some embodiments, the vector comprises a transposase system, such as the PiggyBac transposon system (see Examples). The PiggyBac transposon is a TTAA-specific mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut-and-paste" mechanism. The PiggyBac transposase recognizes transposon-specific inverted terminal repeats (ITRs) and moves the intervening contents to a TTAA insertion site in a chromosome or another vector. Thus, inserting a gene of interest between two ITRs in a transposon-based vector allows for efficient insertion of the gene into the target genome. Other suitable transposase systems for use in the present invention include, for example, Sleeping Beauty. In other embodiments, the vector is a plasmid, a viral vector, a cosmid, or an artificial chromosome. Suitable plasmids include, for example, E. coli cloning vectors. Many suitable viral vectors are known in the art, including, but not limited to, adenovirus vectors; adeno-associated virus vectors; poxvirus vectors, such as fowlpox virus vectors; alphavirus vectors; baculovirus vectors; herpesvirus vectors; retrovirus vectors, such as lentivirus vectors; modified vaccinia virus Ankara vectors; Ross River virus vectors; Sindbis virus vectors; Semliki Forest virus vectors; and Venezuelan equine encephalitis virus vectors. In a specific embodiment, the vector comprises SEQ ID NO: 15.

[0025] In some embodiments, the vector is an expression vector comprising a promoter that drives expression of the SOX18 transgene, preferably a transient or inducible expression of the SOX18 transgene. As used herein, the term "promoter" refers to a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a coding sequence. While promoters are most commonly found immediately upstream of a coding sequence, they can also be found downstream of or within a coding sequence. Promoters may be derived entirely from a native gene or may be composed of multiple elements, including elements derived from naturally occurring promoters or elements containing synthetic DNA sequences. It is understood by those skilled in the art that different promoters can direct gene expression in different tissues or cell types, at different stages of development, or in response to different environmental conditions. Preferably, the promoter used in the present invention is an inducible promoter. An "inducible promoter" is a promoter that is activated (i.e., initiates transcription) only in the presence of a specific molecule. Inducible promoters allow for tight regulation of transgene expression in cells. Numerous suitable inducible expression systems are well known in the art, including, for example, the Tet-On gene expression system, in which gene expression can be induced by administering tetracycline (Tc) or a tetracycline derivative like doxycycline (DOX). Suitable Tet-On systems for use in the present invention include, but are not limited to, improved Tet-On and Tet-On 3G. The Tet-On system utilizes several promoters, including both minimal promoters (e.g., CMV) flanked by tetracycline-responsive elements (TREs) and engineered Tet-inducible promoters (e.g., TRE2 and TREtight). For example, in the examples, the SOX18 transgene is inserted into a vector downstream of the doxycycline-inducible TREtight promoter (i.e., using conventional cloning methods).This vector is introduced into hPSCs, allowing us to induce expression of the SOX18 transgene at the desired stage of differentiation by adding doxycycline to the cell culture to activate expression from the TREtight promoter. Those skilled in the art will recognize numerous additional inducible gene expression systems, including both chemically and temperature-inducible systems. Other suitable inducible gene expression systems for use in the present invention include, but are not limited to, the glucocorticoid-responsive mouse mammary tumor virus promoter (MMTVprom), the tamoxifen-responsive hormone-binding domain of the estrogen receptor (ERTAM), the ecdysone-inducible promoter (EcP), heat-shock-inducible promoters (e.g., promoters derived from Hsp70 or Hsp90), and the T7 promoter / T7 RNA polymerase system (T7P). The SOX18 transgene can be introduced into hPSCs using any suitable method, for example, by transfection or transduction. In one embodiment, the transgene is introduced by transducing hPSCs with a vector containing the SOX18 transgene. In another embodiment, hPSCs are transduced with exogenous SOX18 modified mRNA (mmRNA). In yet another embodiment, hPSCs are transduced with SOX18 protein. Typically, the mmRNA contains (i) a 5' synthetic cap for enhanced translation; (ii) modified nucleotides that confer RNAse resistance and attenuate cellular interferon responses that otherwise greatly reduce translation efficiency; and (iii) a 3' polyA tail.

[0026] As provided herein, SOX18 can be forced in progenitor cells by the introduction of an inducible recombinant genetic construct encoding human SOX18 having a nucleotide sequence described herein. As used herein, the term "force," when used in reference to SOX18 expression in progenitor cells for NK cells or megakaryocytes produced by the methods disclosed herein, can be understood by those skilled in the art to mean that SOX18 expression in the cells is increased above endogenous SOX18 expression by the introduction of an exogenous SOX18-encoding construct, wherein SOX18 expression is regulated by an inducible promoter. Endogenous SOX18 can be forced using reagents and methods well known in the art, including, but not limited to, those disclosed in U.S. Patent Application Nos. 2018 / 0010124 and 2018 / 0142207, the disclosures of which are expressly incorporated herein by reference. Such techniques and methods can include forced expression of SOX18 in the cells.

[0027] As used herein, the term "forced expression" refers to inducing increased levels of a protein of interest (e.g., a transcription factor) in a host cell, such as a population of hPSCs. Forced expression can include any combination of one or more of the following: introducing an exogenous nucleic acid encoding the protein of interest (e.g., by viral transduction, plasmid expression vector transfection, or variable mRNA transfection); protein transduction; genomic modification of the host cell, such as replacing a promoter to increase expression of an endogenous (native) gene; and contacting the host cell with a small molecule that induces increased expression of the endogenous protein.

[0028] Pharmaceutical Formulations and Methods of Treatment Administration of a therapeutically effective amount of NK cells or megakaryocytes provided herein to a recipient subject is generally accomplished using methods well known in the art and typically involves introducing a therapeutically effective dose of NK progenitor cells into a subject using direct injection or other clinical procedures well known to those skilled in the art (e.g., U.S. Patent Nos. 6,447,765; 6,383,481; 6,143,292; and 6,326,198). For example, introduction of the NK cells or megakaryocytes of the present invention can be locally or systemically injected via intravascular administration, e.g., intravenous, intramuscular, or intraarterial administration, intraperitoneal administration, etc. The cells can be injected into an infusion bag (e.g., a Fenwal infusion bag (Fenwal, Inc.)) using a sterile syringe or other sterile transfer device. The cells can then be immediately infused via IV administration into a free-flowing IV line into the patient for a period of time, e.g., 15 minutes. In some embodiments, additional reagents, such as buffers or salts, are provided to the recipient subject simultaneously with the cells. Various illustrative embodiments of the compositions and methods according to the present invention are described herein in the following non-limiting examples. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and following embodiments and are within the scope of the appended claims. [Example]

[0029] The examples described herein incorporate and rely on certain experimental and preliminary methods and techniques, which are performed as illustrated herein. Materials and Methods Cell Culture Wild-type H1 and human pluripotent stem cells (hPSCs) were obtained from WiCell (Madison, WI). Vector Construction, Generation, and Validation of iSOX18 hPSC Lines. A doxycycline (DOX)-inducible SOX18 H1 hESC line was generated using the PiggyBac system described in Park et al., 2018, Curr. Protoc. Stem Cell Biol. 47: e63, and illustrated in Figure 1. The human SOX18 CDS was cloned downstream of the TREtight promoter in the PiggyBac transposon vector (Transposagen), pTRE-P2A-Venus-rpEF1a-Zeo plasmid, and cotransfected with pEF1α-M2rtTA-T2A-Puro and the transposase plasmid into H1 cells using the Human Stem Cell Nucleofector Kit 2 (Lonza). Cells were selected in Zeocin (0.5 μg / ml, Thermofisher) and puromycin (0.5 μg / ml, Sigma) for 10 days, and resistant clones were screened by Venus expression with DOX (Sigma) treatment.

[0030] hPSC Maintenance and Hematopoietic Differentiation hPSCs were maintained and passaged on Matrigel in mTeSR1 medium (WiCell). Hematopoietic differentiation was performed on collagen IV (ColIV)-coated plates in defined serum-free medium as previously described (Uenishi et al., 2014, Stem Cell Reports 3: 1073-1084). The hPSC-derived iSOX18 H1 line (H1 hESC line from WiCell) was maintained and passaged on Matrigel in mTeSR1 medium (WiCell). Cell lines were differentiated on collagen IV (ColIV)-coated plates. Briefly, cells were plated at 5,000 cells / cm to initiate differentiation. 2Cells were plated in 6-well plates containing E8 medium and 10 μM Rock inhibitor (Y-27632, Cayman Chemicals) at 10 °C. The next day, the medium was replaced with IF9S medium containing 50 ng / ml FGF2 (PeproTech), 50 ng / ml BMP4 (PeproTech), 15 ng / ml activin A (PeproTech), and 2 mM LiCl (Sigma), and the cells were cultured under hypoxia (5% CO2, 5% O2). On day 2, the medium was replaced with IF9S medium containing 50 ng / ml FGF2, 50 ng / ml VEGF (PeproTech), and 2.5 μM TGF-β inhibitor (SB-431542, Cayman), and the cells were cultured under hypoxia (5% CO2, 5% O2). On days 4 and 6, the medium was replaced with IF9S medium containing 50 ng / ml FGF2, 50 ng / ml VEGF, 50 ng / ml TPO (PeproTech), 50 ng / ml IL-6 (PeproTech), 20 ng / ml SCF (PeproTech), and 10 ng / ml IL-3 (PeproTech), and the cells were cultured under normoxia (20% CO2, 5% O2).

[0031] Isolation and culture of hemogenic endothelial cells and hematopoietic progenitor cells. CD31 cells were isolated from day 4 (D4) cultures of iSOX18 cells. + Cells were isolated by magnetic-activated cell sorting (MACS) using CD31 antibodies. In D4 differentiated cultures, almost all CD31 +The cells co-express VE-cadherin. Selected cells were plated on OP9 or DLL4-OP9 in 10% α-MEM containing 10% FBS (Hyclone) and TPO, SCF (50 ng / ml), IL-6 (20 ng / ml), IL-3, and FLT3L (10 ng / ml). The medium was changed the next day, and additional medium was added to the OP9 co-cultures on day 3. Five days after subculture, cells were harvested and evaluated for colony-forming cell (CFC), T, and NK differentiation potential. Hematopoietic progenitor cells (HP) generated from iSOX18 cells were harvested on day 8 (D8) of differentiation. Four distinct subsets, namely, the CD34+CD43+ population, CD235a / CD41a-CD45- (P1 subset), CD235a / CD41a+CD45- (P2), CD235a / CD41a+CD45+ (P3), and CD235a / CD41a-CD45+ (P4), were isolated using an MA900 cell sorter (Sony Biotechnology) and cultured on OP9-DLL4 cells under NK cell differentiation conditions.

[0032] Hemangioblast (HB)-CFC and hematopoietic CFC assays. HB-CFCs were detected as described in Vodyanik et al., 2010, A mesoderm-derived precursor for mesenchymal stem and endothelial cells, Cell Stem Cell 7: 718-729. Hematopoietic CFCs were detected using serum containing H4435 MethoCult (Stem Cell Technologies). HB-CFCs were detected using semi-fluid colony-forming serum-free medium (CF-SFM) containing 40% ES-Cult M3120 methylcellulose (2.5% solution in IMDM, Stem Cell Technologies), 25% StemSpan serum-free growth medium (SFEM, Stem Cell Technologies), 25% human endothelial cell serum-free medium (ESFM, ThermoFisher), 10% BIT 9500 supplement (Stem Cell Technologies), GlutaMAX (1 / 100 dilution, ThermoFisher), Ex-Cyte (1 / 1000 dilution, Millipore), 100 mM MTG, 50 mg / mL ascorbic acid, and 20 ng / mL FGF (Peprotech).

[0033] Megakaryocytic differentiation: Suspension hematopoietic cells from iSOX18 hPSC cultures were collected on day 8 of differentiation and cultured for 5 days on ultra-low attachment 6-well plates in StemSpan serum-free growth medium (SFEM, Stem Cell Technologies) containing 20 ng / ml SCF, TPO, and IL-11. Fresh medium (2 mL) was added every 2 days. All cytokines were purchased from PeproTech. NK Cell Differentiation. For NK cell differentiation, suspension hematopoietic cells from D8 primary cultures or day 4+5 secondary OP9 / OP9-DLL4 cocultures were cultured on OP9-DLL4 for 5 days in α-MEM (Invitrogen) containing 20% ​​FBS (Hyclone), 25 ng / ml SCF, 5 ng / ml FLT3L, IL-3 and IL-7, and 10 ng / ml IL-15 (PeproTech). Cells were then cultured in the same medium without IL-3 for 3–4 weeks. Cells were passaged weekly onto fresh DLL4-OP9 cells and analyzed by flow cytometry for NK cell surface markers after 3–4 weeks.

[0034] Functional analysis of NK cells. To assess cytotoxicity, CD56 + Cells were isolated using an MA900 cell sorter and incubated with K562-GFP target cells for 4 hours at 37°C at effector:target (E:T) ratios of 1:1, 2.5:1, and 5:1 in 96-well plates. Cells were collected in FACS buffer and stained with 7-aminoactinomycin D (7-AAD) and annexin V (BD). Specific killing was calculated by subtracting spontaneous K562 death (7-AAD+ cells in the effector-free control). Isolated CD56 + IFNγ production and CD107a expression in cells were assessed after 4 hours of incubation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (1:500) (BioLegend). Brefeldin A (1:1000; ThermoFisher) was added at the beginning of stimulation. Cells were washed with FACS buffer and stained with Live / Dead Violet 540 (TONBO Bioscience) together with CD107a antibody (BD). Cells were treated with fixation / permeabilization buffer (eBioscience) and stained for intracellular IFNγ (BD).

[0035] For T cell differentiation, suspension hematopoietic cells from day 8 of primary differentiation cultures or day 4+5 secondary OP9 / OP9-DLL4 cocultures were cultured on OP9-DLL4 in α-MEM (Invitrogen) containing 20% ​​FBS (Hyclone), 10 ng / ml SCF, 5 ng / ml FLT3L, and IL-7 (PeproTech) for 3 weeks. Cells were passaged weekly onto fresh OP9-DLL4 cells. Cells were analyzed by flow cytometry for T cell surface markers after 21 days. All cytokines were purchased from PeproTech. Flow cytometry and t-distributed stochastic neighbor embedding algorithm (tSNE) analysis. Flow cytometry analysis was performed using the antibodies listed in Table 1 using a MACSQuant Analyzer 10 (Miltenyi Biotech) and FlowJo software (FlowJo LLC). For tSNE analysis, individual DOX2-8 and DOX-free fcs files were imported into FlowJo, and doublets, debris, and dead cells were removed. The CD43 subset or CD43 + 11,000 cells were selected for each sample and concatenated. To generate tSNE maps, the concatenated data were analyzed using the following parameters: perplexity 30, iteration number 550, and learning rate (Eta) 1540. Concatenated cells were manually divided into no DOX or DOX2-8, and cell subsets were determined by manual gating. First, cells CD34 + or CD34 - Gating on CD235a / CD41a hi CD45 - , CD235a / CD41a med CD45 - , CD235a / CD41a - CD45-, CD235a / CD41a - CD45 + or CD235a / CD41a + CD45 + Gating was performed on cells.

[0036] Apoptosis and cell cycle analysis. Apoptosis was detected by flow cytometry using Annexin V (BD). For cell cycle analysis, D5 cells were incubated in culture medium containing bromodeoxyuridine (BrdU) (10 μM, BD Pharmingen) for 2 hours and stained with antibodies. For BrdU detection, a BrdU flow kit with 7AAD was used according to the manufacturer's instructions. The fluorescent reagents used for analysis, cell viability, apoptosis, and proliferation are listed in Table 2. Real-time quantitative polymerase chain reaction (qPCR) RNA was isolated from control and DOX-treated cultures of iSOX18, iSOX173 hPSCs using the RNeasy Plus Micro Kit (QIAGEN). + RNA was extracted from cells. RNA was reverse transcribed into cDNA using random hexamer primers (QIAGEN) with SMART MMLV reverse transcriptase (TaKaRa). qPCR was performed using TB Green Advantage qPCR Premix (TaKaRa). RPL13A was used as a reference gene to normalize the data. Primer sequences are listed in Table 2.

[0037] RNA-Seq Data Processing and Analysis: One hundred nanograms of total RNA was used to prepare sequencing libraries following ligation-mediated sequencing (LM-Seq) protocol (see Gandillet et al., 2009, Blood 114: 4813-422) and quantification using a Qubit fluorometer (Life Technologies). The final cDNA libraries were quantified using the Quant-iT PicoGreen Assay Kit (ThermoFisher Scientific), multiplexed, loaded at a final concentration of 1 nM or 2.5 nM, and sequenced as single reads on a NextSeq 2000 (Illumina). RNA-seq reads were aligned to the human genome (version hg38) using GENCODE basal gene annotations (version 38) using STAR (version 2.5.2b). Gene expression levels were quantified using RSEM (version 1.3.0), and differential expression was analyzed using edgeR (version 3.34.1). Differentially expressed genes were required to have at least a 2-fold change and an adjusted P value <0.05. Gene set enrichment analysis was performed using fgsea (version 1.18.0) using KEGG gene sets from the Molecular Signatures Database (version 7.1). RNA-seq data were deposited in GEO under the accession code GSE195670 and review token yfefeuuwtvcfncx.

[0038] Western Blot For Western blot experiments, iSOX18 hPSCs were cultured with and without DOX (2 μg / ml) for 24 hours and then harvested. In a similar manner, all cells from iSOX18 hPSC differentiation cultures with and without DOX (D2–D5) were harvested on day 5 of differentiation. Cells were solubilized using Pierce IP Lysis Buffer containing Pierce protease inhibitors. For cell lysate analysis, protein levels were quantified using the Pierce BCA Assay kit (Thermo Fisher, Waltham, MA) and normalized to 8 μg total protein (dependent on individual blots). Precast 4–12% gradient SDS-PAGE gels were run and subsequently transferred to PVDF membranes using a Bio-Rad Trans-Blot Turbo Transfer System. The membranes were blocked with 5% BSA (Fisher Scientific, BP1600-100) and 5% Difco skim milk (BD, 232100) in TBST (1%) for probing with human SOX18 antibody (R&D Systems, 1:1000) and anti-GAPDH (Santa Cruz Biotechnology, 1:5000), respectively. After blocking, the membranes were incubated with the primary antibodies overnight at 4°C with gentle agitation. The membranes were blotted with their corresponding HRP-conjugated secondary antibodies for 1 hour at room temperature. The antibodies were diluted in 1% BSA and 1% milk in TBST for SOX18 and GAPDH detection, respectively. 1% TBST was used to wash the membranes three times, with 5-minute intervals. Sheep and rabbit HRP-conjugated secondary antibodies were purchased from R&D Systems and Santa Cruz Biotechnology, respectively. Images were captured using a Bio-Rad ChemiDoc XRS+.

[0039] Statistical Analysis Data were analyzed using GraphPad Prism version 9 (GraphPad Software Inc.) and Microsoft Excel (Microsoft Corporation). Tests for statistical significance are listed for each experiment; these include two-tailed Student's t-tests for paired analyses, and one-way and two-way ANOVAs with Tukey and Sidak post-hoc tests for experiments involving multiple comparisons of variables or grouped variables, as estimated most appropriately by the software.

[0040] Example 1 Forced expression of SOX18 enhances the production of erythromyeloid progenitors. To determine the impact of SOX18 overexpression on hematopoietic development in humans, we generated H1 hESCs carrying doxycycline (DOX)-inducible SOX18-P2A-Venus (Figs. 1 and 7) and differentiated these cells in a synthetic culture system in which all stages of hematopoietic development were temporally, phenotypically, and functionally defined, as further described in Example 2 (Fig. 2A).

[0041] To determine the stage of hematopoietic development sensitive to SOX18 regulation and the optimal duration of SOX18 overexpression to achieve the maximum effect on hematopoietic outcome, hESC differentiation cultures were treated with DOX for various time points, and the phenotype and colony-forming cell (CFC) differentiation potential of hematopoietic cells harvested on differentiation day 8 (D8) were analyzed (Figure 2B). As shown in Figures 2C-2D, treatment of cultures with DOX from day 2 (D2) to D8 (DOX2-8) significantly increased the CD34 + CD43 + This treatment resulted in the highest percentage of HP and CFC. + CD43 + CD235a / CD41 in cell populations - CD45 - It also increased the proportion of progenitor cells (D8 P1 population), and CD235a / CD41a + CD45 +DOX treatment at this stage of differentiation had a modest effect on the percentage of CD34+CD43+ cells in the present study, although a similar effect on subset composition within CD34+CD43+ progenitor cells was observed in DOX6-8 and, to a lesser extent, in DOX4-6 cultures. In contrast, early, short-term treatment (DOX2-4) modestly increased CD34+CD43+ cells but had a modest effect on their composition. These findings suggested that SOX18 has its most profound effect on hematopoietic differentiation when sustainedly upregulated from D2 to D8 of differentiation. Analysis of colony-forming unit (CFC) differentiation potential demonstrated that SOX18 contributes to the differentiation of GM cells. - We found that SOX18 overexpression had the most pronounced effect on erythroblastic and E-CFCs. This was most evident in DOX2-6 and DOX2-8 cultures (Figure 2E). DOX-treated cultures also had a higher megakaryocytic differentiation potential than untreated controls (Figure 9A). These observations indicated that SOX18 overexpression primarily promoted erythromyeloid progenitors.

[0042] To visualize the detailed phenotype of D8 hematopoietic cells from DOX-free and DOX2-8 cultures, CD43+-gated cells were analyzed using a t-distributed stochastic neighbor embedding algorithm (tSNE), which showed different single-cell deposition between DOX-free and DOX2-8 cultures (Figure 8A). tSNE analysis revealed that CD45+ cells were significantly different in CD45+ cells after DOX treatment. + Population suppression and CD45 - and CD235a / 41a - Additionally, DOX treatment enhanced the enrichment of the CD235a / 41a population, which corresponds to more mature erythromyeloid progenitors. hi / med CD34 - The population with the phenotype was reduced (Figures 8B-8C). - and consistent with previous findings in mouse systems that demonstrated the effect of blocking Sox18 on the differentiation of yolk sac-derived HPs (see Serrano et al., 2010, Blood 115: 3895-3898).

[0043] Example 2 Forced expression of SOX18 promotes megakaryocyte production. To determine the effects of SOX18 overexpression on hematopoietic development in humans, we generated H1 hESCs carrying doxycycline (DOX)-inducible SOX18-P2A-Venus (Figures 1 and 7) and differentiated these cells in a synthetic culture system in which all stages of hematopoietic development were temporally, phenotypically, and functionally characterized. In this differentiation system, the most primitive hematopoietic cells, capable of FGF2-dependent hematohemangioblast colony-forming cell (HB-CFC) differentiation, are detected at day 3 (D3) of differentiation. The first immature / primitive VECs express high levels of the HAND1 mesoderm gene and lack arterial and venous gene expression. + CD43 - CD73 - NOTCH1 + HE cells arise at D4 (D4 HE). Subsequently, at D5, HE cells give rise to DLL4 cells with distinct lymphoid-myeloid differentiation potential. + CXCR4 + / - DLL4 with arterial HE type and bone marrow-restricted differentiation potential - This results in non-arterial HE type (Figure 2A). + CXCR4 + HE is highly enriched for lymphoid-myeloid progenitors and expresses other DLL4 + and DLL4 - Compared to the HE population, they express the highest levels of HOXA and arterial genes, including SOX17 and NOTCH4. All hematopoietic progenitor cells from hPSC cultures at D8 of differentiation can be identified by CD43 expression (Vodyanik et al., Blood 108:2095-2105 (2006); Choi et al., Stem Cell 27:559-567 (2009)). D8 CD34 + CD43 + HP consists of at least three major subpopulations: (1) CD235a-enriched erythro-megakaryocytic cells; + CD41a + CD45 - progenitor cells, (2) CD41a with erythromyeloid differentiation potential; lo CD235a+ / - CD45 + progenitor cells, and (3) lacking lineage markers, such as CD90, typical of human hematopoietic stem / progenitor cells (HPSCs) Ivanovs et al., Stem Cell Rep. 2:449-456 (2014). + CD38 - CD45RA - CD235a presenting phenotype - CD41a - CD43 + CD45 + / - Multilineage progenitor cells (Vodyanik et al., Blood 108:2095-2105 (2006); Choi et al., Stem Cell 27:559-567 (2009); Suknuntha et al. Stem Cell Res. 15:678-693 (2015); Mesquitta et al. Sci. Rep. 9:6622 (2019)). In addition, SOX18 expression was shown to initiate at D4 differentiation in HE and remain present at D5 HE and D8 HP.

[0044] To determine the stage of hematopoietic development sensitive to SOX18 regulation and the optimal duration of SOX18 overexpression to achieve the maximum effect on hematopoietic outcome, hESC differentiation cultures were treated with DOX at various time points, and the phenotype and CFC differentiation potential of hematopoietic cells collected on D8 of differentiation were analyzed (Figure 2B). As shown in Figures 2C-2D, treatment of cultures with DOX from D2 to D8 (DOX2-8) significantly increased the CD34 + CD43 + This treatment resulted in the highest percentage of HP and CFC. + CD43 + CD235a / CD41 in cell populations - CD45 - Increased the proportion of progenitor cells (D8 P1 population) and CD235a / CD41a + CD45 +DOX treatment at these stages of differentiation had a modest effect on the percentage of CD34+CD43+ cells, although a similar effect was observed in DOX6-8, and to a lesser extent in DOX4-6 cultures, on the subset composition within CD34+CD43+ progenitor cells. In contrast, early, short-term treatment (DOX2-4) modestly increased CD34+CD43+ cells but had a modest effect on their composition. These findings suggest that SOX18, when persistently upregulated from D2 to D8 of differentiation, may be involved in the regulation of CD34+CD43+ cells. + CD43 + It was suggested that HP and CFC had the most significant effect. Analysis of CFC differentiation potential revealed that SOX18 had the most pronounced effect on GM- and E-CFCs. This was most evident in DOX2-6 and DOX2-8 cultures (Figure 2E). Furthermore, we found that DOX-treated cultures had even greater megakaryocytic differentiation potential than untreated controls (Figures 9A-9B). These observations indicate that SOX18 overexpression, starting at D2 of differentiation, primarily promotes erythromyeloid progenitors.

[0045] Example 3 Forced expression of SOX18 suppresses T cell differentiation and promotes NK cell production. To assess the effect of SOX18 on lymphoid differentiation, cultures were treated with DOX as shown in Figure 2B, and the T cell and NK cell differentiation potential of HP generated on day 8 of 9-day differentiation was assessed. Treatment of differentiation cultures with DOX suppressed the T cell differentiation potential of day 8 HP. This suppression of T cell differentiation potential was even more pronounced in cultures treated with DOX from D2 to D8 (Figures 2F-2G). However, HP from DOX-treated cultures retained CD56 + These DOX treatments were able to generate NK cells, the production of which increased most dramatically after prolonged SOX18 upregulation during hematopoietic differentiation (DOX2-6 and DOX2-8 cultures; Figures 2H-2I). +It also increased CD16 expression by the cells, whereas CD94 expression increased between D2 and 4 or D2 and 6 but decreased during D2 and 8 DOX treatment. Overall, these findings suggest that forced expression of SOX18 between days 2 and 8 (D2 and 8) of differentiation promoted NK cell development and slightly affected CD16 and CD94 expression.

[0046] Example 4 Forced expression of SOX18 promoted HB-CFC but had limited effect on the specification of hemogenic endothelial cells. To determine the stage of hematopoietic development primarily affected by SOX18 overexpression, we analyzed the effects of DOX treatment on the formation of hemangioblast (HB) colonies and HE, including arterial HE specification. Forced expression of SOX18 at D2 of differentiation resulted in a nearly three-fold increase in the number of HB colonies (Figure 9B). However, SOX18 overexpression had limited effects on HE formation and arterial HE specification at D4 and D5 of differentiation (Figures 3A-3F). VE-cadherin expression in DOX-treated cultures was significantly increased. + (VEC + Although a slight increase in the proportion of endothelial cells was observed, DLL4 was significantly increased in both DOX and DOX-free cultures. + CXCR4 + / - No significant differences in the rate of arterial HE were observed. - CXCR4 + Cluster formation was also observed. - CXCR4 + and DLL4 - CXCR4 - D4 VEC + Transcriptional profiling of the population did not reveal any differentially expressed genes (as described below).

[0047] To evaluate the effect of SOX18 on HE, D4 HE generated under DOX-free and DOX conditions were isolated and cultured on OP9 or OP9-DLL4 in the presence or absence of DOX (Figure 4A). Analysis of CFC differentiation potential revealed that DOX treatment had the most significant effect on HE cultured on OP9-DLL4. In these cocultures, sustained treatment with DOX significantly increased CFC formation, whereas HE derived from primary differentiation cultures pretreated with DOX (DOX2-4) and without additional DOX treatment during coculture generated fewer CFCs compared to the no-DOX control (Figure 4B).

[0048] Analysis of T cell differentiation potential revealed that HE from DOX2-4 treated cultures demonstrated a decrease in total T cell output when cultured on OP9 and OP9-DLL4. This effect was consistent with the results of DOX treatment on OP9 or OP9-DLL4. - This was even more evident in cocultures of HE with DLL4 initiated on D4 or in DOX2-8-treated cultures (Figures 4C-4D). In contrast, treatment of HE with OP9 or OP9-DLL4 cultures significantly increased the NK cell differentiation potential of D8 HP, whereas D2-4 DOX treatment of primary differentiation cultures had no effect on NK cells (Figures 4E-4F). These findings suggested that SOX18 overexpression primarily affects lymphocyte specification from HE by shifting the balance of NK versus T lymphocyte differentiation potential.

[0049] Example 5 Forced expression of SOX18 promotes the expansion of the CD34+CD43+CD235a / CD41a-CD45-(D8 P1) population, which is enriched for NK cell differentiation. As shown in Figures 2C and 2D, D2-D8 SOX18 overexpression was associated with CD235a / CD41a - CD45 - (D8 P1) significantly promoted the development of the CD34 + CD43 + CD235A / CD41a in HP + (D8 P3) and CD235a / CD41a -(D8 P4) population, including CD45 + To determine the cell population enriched for NK cells after DOX treatment, the main population of HPs formed in the D8 cultures was isolated and their NK cell differentiation potential was assessed (Figure 6A). As shown in Figures 6B and 6C, in the control cultures, NK cell differentiation potential was mainly detected in the D8 P1 and P4 populations, while the P2 population failed to produce NK cells. CD235a / CD41a + CD45 + Although (D8 P3) cells are capable of producing NK cells, the total NK cell production from this subset was negligible compared to the P1 and P4 populations. + Due to the dramatic inhibition of cell development, we evaluated the NK cell differentiation potential of only two major populations: i) the D8 P1 population, which expanded significantly after DOX treatment, and ii) the D8 P2 population. These studies revealed that in DOX-treated cultures, NK cell differentiation potential was limited to the D8 P1 population, which possessed even stronger NK cell differentiation potential compared to the NK-producing population in control, DOX-free cultures (Figures 5B and 5C). Overall, HPs recovered from DOX2-8-treated cultures expanded more rapidly and for longer periods in NK cell differentiation cultures and were able to generate fivefold more NK cells compared to controls (Figure 5D). No differences were observed in the cytotoxic differentiation potential of NK cells generated from DOX+ and DOX- conditions against K562 cells (Figure 5E). However, in response to PMA, NK cells from DOX-treated cultures displayed robust IFNg production and a slightly reduced degranulation response compared to NK cells from untreated cultures (Figures 5F and 5G). Overall, these studies demonstrate that forced expression of SOX18 enhances CD34 expression, which has superior NK cell differentiation potential. + CD43 + CD235a / CD41a - CD45 - (D8 P1) shows that the HP population is predominantly proliferating.

[0050] Example 6 Molecular characterization of SOX18-induced changes in HE and blood cells To clarify the changes in the transcriptional program induced by SOX18 forced expression, RNA-seq analysis was performed on D4 HE, D8 CD34+CD43+ subsets, and NK cells generated from DOX-free and DOX-treated cultures (Figure 6A). After D2-DOX treatment, only 15 differentially expressed genes (DEGs) were detected in the D4 DLL4-CXCR4-VEC+ population (D4 P1 population) (Figure 6B), suggesting a minimal effect of SOX18 on D4 HE. In contrast to previous findings with D2-4 SOX17 overexpression (see Jung et al., 2021, Cell Rep. 34: 108758), RNA-seq analysis of SOX18 D4 HE from DOX-free and DOX cultures did not show a significant increase in the expression of HOXA genes, CDX2, or genes involved in the NOTCH signaling pathway (Figure 6C). These findings were confirmed using qPCR analysis of selected genes in D4 HE cells isolated from control and D2-4 DOX-treated differentiation cultures of iSOX17 and iSOX18 hPSCs (Figure 6D). Because flow cytometry analysis indicated that SOX18 overexpression induced a DLL4-CXCR4+ subset within D4 HE (Figures 3A and 6A), DEGs were analyzed in the DLL4-CXCR4+ (D4 P1) and DLL4-CXCR4- (D4 P2) populations from DOX-treated cultures. These studies revealed no DEGs in these subsets that contained expression of arterial genes, suggesting that CXCR4 expression in D4 HE without coexpression of DLL4 does not reflect activation of the arterial program. The most striking changes in the transcriptional program were observed in CD56+ NK cells, which displayed 209 and 811 DEGs in the D8 P1 multipotent HP population and their corresponding DOX-treated and untreated cultures (Figure 6B).Gene set enrichment analysis (GSEA) in D8 P1 cells revealed enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG) categories related to T cell receptor (TCR) and Toll-like receptor (TLR) signaling pathways (Figure 6E), suggesting that SOX18 overexpression has a primary impact on establishing the T lymphoid transcriptional program in D8 P1 multipotent HPs emerging from HE. As shown in Figure 6F, downregulated genes in these categories included CD3 complex genes, CD4, AP1 complex genes JUN and FOS, PI3 signaling genes, and most TLR genes, while upregulated genes included IFNG, TICAM2, TLR5, and TLR9. The D8 P2 populations in the DOX+ and DOX- conditions showed enrichment of only 79 DEGs in the extracellular matrix (ECM) receptor interaction KEGG category (Figure 6E). GSEA of NK cells generated from DOX-treated and untreated cultures demonstrated enrichment in KEGG categories related to glyoxylate and dicarboxylate metabolism, the one-carbon pool by folate, and the PPAR signaling pathway (Figure 6E). However, no differential expression of the NK cell-activating receptors KLRK1, NCR2, and NCR1 or the key NK cell transcription factors EOMES and TBX21 was observed between NK cells from DOX-treated and untreated cultures (Data S1). These findings indicated that overexpression of SOX18 during the endothelial-to-hematopoietic transition (EHT) has a profound effect on the transcriptional program controlling NK cell metabolism.

[0051] To determine whether SOX18 overexpression affected proliferation and apoptosis in major cell subsets, gene set enrichment analysis was performed on GO categories related to "regulation of cell population proliferation" and "apoptosis." The first GO gene set significantly changed in the D4 and D8 P1 subsets and NK cells in the DOX+ vs. DOX-free comparison, while apoptosis genes primarily affected the D8 P1 subset (Figures 10A and 10B). To confirm the effect of SOX18 on cell proliferation, we performed cell cycle analysis of D5-differentiated cells using bromodeoxyuridine (BrdU). Consistent with the gene expression analysis, SOX18 overexpression led to a significant cell cycle shift from G0 / G1 to S phase in HE cells, resulting in the appearance of CD43+HP cells (Figures 10C and 10D).

[0052] [Table 1]

[0053] [Table 2]

[0054] 22-0629-US-PRO_Sequence-Listing_ST25 1. PiggyBack-SOX18 construct sequence (SEQ ID NO: 15) 1A. Human SOX18 CDS (ORF) (SEQ ID NO: 16) *** ***=start (ATG) codon SOX18 amino acid sequence (SEQ ID NO: 17) MQRSPPGYGAQDDPPARRDCAWAPGHGAAADTRGLAAGPAALAAPAAPASPPSPQRSPPRSPEPGRYGLSPAGRGERQAADESRIRRPMNAFMVWAKDERKRLAQQNPDLHNAVLSKMLGKAWKELNAAEKRPFVEEAERLRVQHLRDHPNYKYRPRRKKQARKARRLEPGLLLPGLAPPQPPPEPFPAASG SARAFRELPPLGAEFDGLGLPTPERSPLDGLEPGEAAFFPPPAAPEDCALRPFRAPYAPTELSRDPGGCYGAPLAEALRTAPPAAPLAGLYYGTLGTPGPPYPGPLSPPPEAPPLESAEPLGPAADLWADVDLTEFDQYLNCSRTRPDAPGLPYHVALAKLGPRAMSCPEESSLISALSDASSAVYYSACISG

[0055] 2. PB-M2rtTA sequence (control element for DOX-inducible SOX18 expression) (SEQ ID NO: 18)

[0056] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. While some embodiments have been illustrated and described in detail in the accompanying drawings and the foregoing description, such illustration and description are considered exemplary and not limiting. Other variations to the disclosed embodiments will be understood from a study of the disclosed drawings and the appended claims, and may arise from practicing the claims. The recitation of certain measurements or characteristics in mutually different dependent claims does not indicate that combinations of these measurements or characteristics cannot be used. Any reference signs in the claims should not be construed as limiting the scope.

Claims

1. A method for increasing stem cell differentiation that produces erythroblastic myelocyte progenitor cells, megakaryocytes, and NK cells, but does not produce differentiated T cells, including forcing SOX18 expression in hematopoietic endothelial cells during the transition from endothelial cells to hematopoietic cell progenitor cells.

2. The method according to claim 1, wherein the cells produced are enriched with NK precursor cells.

3. The method according to claim 2, wherein NK precursor cell production is increased by approximately five times compared to hematopoietic epithelial cells in which SOX18 production is not forced.

4. The method according to claim 1, wherein SOX18 expression is forced after the introduction of an inducible SOX18 coding expression construct.

5. The method according to claim 1, wherein SOX18 expression is forced after the introduction of a constitutively expressed SOX18 coding expression construct.

6. The method according to claim 1, wherein SOX18 expression is forced by introducing modified mRNA (mmRNA) encoding SOX18 into cells.

7. The method according to claim 4, wherein the inducible SOX18 expression construct is forced upon cells by contacting them with doxycycline.

8. A cell culture of NK progenitor cells produced by the method described in claim 1.

9. A composition comprising a cell culture of NK progenitor cells as described in claim 8.

10. A pharmaceutical composition comprising differentiated NK cells produced from the composition according to claim 9, and pharmaceutically (or therapeutically) acceptable excipients and adjuvants.

11. A pharmaceutical composition for use in immunotherapy, comprising differentiated NK cells produced from the composition according to claim 9, and pharmaceutically (or therapeutically) acceptable excipients and adjuvants.

12. A cell culture of megakaryocyte progenitor cells produced by the method described in claim 1.

13. A composition comprising a cell culture of megakaryocyte progenitor cells as described in claim 12.

14. A pharmaceutical composition comprising differentiated megakaryocyte cells produced from the composition according to claim 13, and pharmaceutically (or therapeutically) acceptable excipients and adjuvants.

15. A pharmaceutical composition for use in immunotherapy, comprising differentiated megakaryocyte cells produced from the composition according to claim 13, and pharmaceutically (or therapeutically) acceptable excipients and adjuvants.

16. A composition comprising a differentiated NK cell population produced by the method described in claim 1.

17. A composition comprising a population of differentiated megakaryocyte cells produced by the method described in claim 1.

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