WO2025097004A1 - Modulation of hematopoietic cells using bcl11b - Google Patents

Abstract

In one aspect, a method of modulating hematopoietic differentiation in a stem cell includes inducing expression of BCL11B in the stem cell and differentiating the stem cell to form a differentiated cell. The stem cell may be an induced pluripotent stem cell (iPSC). The differentiated cell may be a natural killer (NK) cell. The differentiated cell may be a T cell. In another aspect, an engineered cell includes a nucleic acid including a sequence of BCL11B and an inducible promoter, wherein expression of BCL11B is controlled by the inducible promoter.

Description

PCT Patent Application Attorney Docket No.0110.000738WO01 MODULATION OF HEMATOPOIETIC CELLS USING BCL11B GOVERNMENT FUNDING This invention was made with government support under HL155150 awarded by the National Institutes of Health. The government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Serial No. 63/595,491, filed on November 2, 2023, the disclosure of which is incorporated by reference herein in its entirety. SEQUENCE LISTING This application contains a Sequence Listing electronically submitted via EFS-Web to the United States Patent and Trademark Office as an XML file entitled “0110-000738WO01.xml” having a size of 7,899 bytes and created on October 29, 2024. The information contained in the Sequence Listing is incorporated by reference herein. SUMMARY This disclosure describes, in one aspect, a method of modulating hematopoietic differentiation in a stem cell. Generally, the method includes inducing expression of BCL11B in the stem cell and differentiating the stem cell to form a differentiated cell. The method may further include differentiating the stem cell into a lymphoid cell, such as an NK cell or a T cell. In one or more embodiments, the stem cell is an induced pluripotent stem cell (iPSC) or a hematopoietic stem cell (HSC). In one or more embodiments, wherein BCL11B is induced in the stem cell in vitro. The method may further include isolating the differentiated cell. In one or more embodiments, the NK cell is a CD56^bright NK cell. In one or more embodiments, the T cell is a CD8⁺ T cell. In one or more embodiments, expression of BCL11B is induced during the mesoderm stage of hematopoietic differentiation, during NK cell differentiation, or during both the mesoderm stage of hematopoietic differentiation and during NK cell differentiation. In one or more embodiments, inducing expression of BCL11B involves inducing expression and subsequently stopping induction. In one or more embodiments, the method increases formation of arterial hemogenic endothelial (AHE) cells compared to a stem cell in which expression of BCL11B is not induced. In one or more embodiments, the method increases formation of multipotent hematopoietic progenitor (MHP) cells compared to a stem cell in which expression of BCL11B is not induced. In one or more embodiments, the method increases NK cell cytotoxicity compared to an NK cell differentiated from stem cells in which expression of BCL11B is not induced. In another aspect, the present disclosure relates to a nucleic acid including a sequence of the gene BCL11B and an inducible promoter, wherein expression of the gene BCL11B is controlled by the inducible promoter. The nucleic acid may further include a selectable marker. In another aspect, this disclosure describes an mRNA produced by a nucleic acid that includes a sequence of the gene BCL11B and an inducible promoter, wherein the mRNA includes the open reading frame of BCL11B. In another aspect, the present disclosure relates to a gene expression vector including a nucleic acid described herein. In another aspect, the present disclosure relates to an engineered cell including the nucleic acid of any preceding embodiment. The engineered cell may be a stem cell, such as an iPSC or an HSC. In one or more embodiments, the nucleic acid is integrated into the genome of the cell. The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. BRIEF DESCRIPTION OF THE FIGURES FIG.1 shows a schematic representation of an exemplary embodiment of an all-in-one Tet-on 3G inducible system construct and targeted knocked-in strategy at the endogenous AAVS1 safe harbor locus via CRISPR/Cas9-mediated homologous recombination. FIG.2 shows analysis of targeted gene knock-in in bone marrow-derived iPSCs for inducible expression of BCL11B. (A) RT-PCR analysis of BCL11B expression in the indicated clones from iPSC lines treated with or without doxycycline (dox). (B) Karyotypes of wild type iPSCs and genetically engineered iPSCs. FIG.3 shows validation of the 3G inducible VENUS system for transient BCL11B induction. Fluorescent microscope images (top) and flow cytometry plots (bottom) show the time-dependent dynamics of VENUS expression with and without 2 μg/ml doxycycline (dox) treatment. VENUS was used as the fluorescent reporter for monitoring BCL11B expression after induction with doxycycline. FIG.4 shows representative flow cytometry plots showing the dose-dependent dynamics of VENUS expression with a range of doxycycline concentrations. VENUS expression is shown on the y-axis, and FSC is shown on the x-axis. FIG.5 shows stage-specific effects of BCL11B expression on hematopoietic development. Flow cytometry analysis shows the presence of arterial hemogenic endothelium derived from iBCL11B1-hiPSCs treated with doxycycline during the mesoderm stage (bottom) and not treated with doxycycline (top). FIG.6 shows stage-specific effects of BCL11B on hematopoietic development. Flow cytometry analysis shows the presence of multipotent hematopoietic progenitors derived from iBCL11B hiPSCs treated with doxycycline during the mesoderm stage (bottom) and not treated with doxycycline (top). FIG.7 shows elevated expression of surface receptors that regulate NK cell effector function in response to Bcl11b induction. Representative flow cytometry plots show surface expression levels of the indicated receptors on iPSC-derived NK cells treated without doxycycline for the induction of BCL11B during in vitro differentiation. FIG.8 shows elevated expression of surface receptors that regulate NK cell effector function in response to Bcl11b induction. Representative flow cytometry plots show surface expression levels of the indicated receptors on iPSC-derived NK cells treated with doxycycline for the induction of BCL11B during in vitro differentiation. FIG.9 shows generation and validation of a synthetic BCL11B mRNA. (A) Schematics of the structural elements of the BCL11B and mAzami control mRNAs generated for testing the effects of BCL11B overexpression in human NK cells. (B) Bleach gel electrophoresis image of BCL11B and control mAzami mRNAs FIG.10 shows peripheral blood NK cells transfected with control mAzami mRNA or BCL11B mRNA and analyzed by flow cytometry 24 hours later. Cells without electroporation were included as a control. (A) Representative FACS plots for lymphocyte gating, viable cell gating, and mAzami-positive frequencies. (B) Cumulative results. Means and SEMs are shown and represent four NK cells from four donors in two different experiments. FIG.11 shows generation and validation of a synthetic BCL11B mRNA. (A) qRT-PCR analysis of BCL11B mRNA in NK cells 24 hours after transfection. Results are from four donors in two different experiments. (B) Western blot using antibodies against Bcl11b and beta-actin and protein from NK cells 48 hours after transfection with control mAzami mRNA or BCL11B mRNA. FIG.12 shows BCL11B restricts CD56^bright NK cell proliferation. (A) Cumulative results of mAzami expression frequencies in CD56^bright NK cells transfected with control and BCL11B mRNAs at each time point. Data represent mean ^ SEM; *P < 0.05, two-sided t-test. (B) Cumulative CELLTRACE (Life Technologies, Corp., San Diego, CA) data at day 7. Data represent mean ^ SEM; *P < 0.05, **P < 0.01, 2-way ANOVA with multiple comparisons. FIG.13 shows BCL11B slows CD56^dimCD57- NK cell proliferation and enhances maturation. (A) Cumulative results of mAzami expression frequencies in CD56^bright NK cells transfected with control and BCL11B mRNAs at each time point. Data represent mean ^ SEM; *P < 0.05, **P < 0.01, two-sided t-test. (B) Cumulative CELLTRACE (Life Technologies, Corp., San Diego, CA) data from gated CD16-CD57-, CD16⁺CD57-, and CD16⁺CD57⁺ NK cell populations at day 7. Data represent mean ^ SEM; *P < 0.05, **P < 0.01, 2-way ANOVA with multiple comparisons. FIG.14 shows cumulative results of percentages of NK cells from control and BCL11B mRNA cultures expressing CD57 at days 1, 5, and 7. Data represent mean ^ SEM; *P < 0.05, two-sided t-test. FIG.15 shows BCL11B mRNA is maintained at high frequencies in CD16⁺CD57⁺ NK cells. (A) Cumulative results of mAzami expression frequencies in CD16⁺CD57⁺ NK cells transfected with control and BCL11B mRNAs at each time point. Data represent mean ^ SEM. (B) Cumulative CELLTRACE (Life Technologies, Corp., San Diego, CA) data at day 7. Data represent mean ^ SEM. FIG.16 shows BCL11B overexpression is associated with increases in cyclin-dependent kinase inhibitors. Bulk CD3-CD56⁺ NK cells were isolated from donor peripheral blood and transfected with mAzami control or BCL11B mRNA. Transfected cells were cultured for seven days with 1 ng/ml IL-15 and harvested at day 7 for intracellular staining of cyclins and cyclin- dependent kinase inhibitors and flow cytometry analysis. Data is from four donors in two independent experiments. (A) Left: cumulative data showing the frequencies of cells expressing CDKN1A in control and BCL11B mRNA cultures. Right: Percentages of CDKN1A positive NK cells in gated CD16-CD57-, CD16⁺CD57-, and CD16⁺CD57⁺ populations. Positive gating for CDKN1A was based on an isotype control. Data represent mean ^ SEM; *P < 0.05, two-sided t- test. (B) Left: cumulative data showing the frequencies of cells expressing CDK2 in control and BCL11B mRNA cultures. Right: Percentages of CDK2 positive NK cells in gated populations. Positive gating for CDK2 was based on an isotype control. Data represent mean ^ SEM. FIG.17 shows BCL11B overexpression is associated with increases in cyclin-dependent kinase inhibitors. (A) Left: cumulative data showing the frequencies of cells expressing CDKN2C in control and BCL11B mRNA cultures. Right: Percentages of CDKN2C positive NK cells in gated populations. Positive gating for CDKN2A was based on an isotype control. Data represent mean ^ SEM; *P < 0.05, two-sided t-test. (B) Left: cumulative data showing the frequencies of cells expressing CDK4 in control and BCL11B mRNA cultures. Right: Percentages of CDK4 positive NK cells in gated populations. Positive gating for CDK4 was based on an isotype control. Data represent mean ^ SEM. FIG.18 shows that less proliferation in NK cells overexpressing BCL11B is associated with increases in cytotoxic granule components. (A) Bulk CD3-CD56⁺ NK cells were isolated from donor peripheral blood and transfected with mAzami control or BCL11B mRNA. Transfected cells were cultured for seven days with 1 ng/ml IL-15. A control condition without electroporation was included. Top: Representative flow cytometry plots showing the gating of FSC^high and FSC^low populations from each culture. Bottom: Cumulative data showing frequencies of FSC^high cells from each culture. Data represent mean ^ SEM; *P < 0.05, **P < 0.01, two-sided t-test. Data is from six donors in three separate experiments. (B) Histogram plots showing intracellular perforin and granzyme B levels, as determined by flow cytometry, in gated of FSC^high and FSC^low populations from a representative donor. Cumulative perforin and granzyme B mean fluorescence intensity (MFI) data from all six donors. Data represent mean ^ SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 2-way ANOVA with multiple comparisons. FIG.19 shows data from an experiment wherein bulk CD3-CD56⁺ NK cells were isolated from donor peripheral blood, labeled with CELLTRACE (Life Technologies, Corp., San Diego, CA) dye, and transfected and cultured as described above. Top: Cumulative data showing the percentages of non-dividing cells from each culture condition gated by FSC. Data represent mean ^ SEM; ***P < 0.05, two-sided t-test. Middle: Flow cytometry plots of CELLTRACE histograms from cells in each culture condition gated by FSC from a representative donor. Bottom: Cumulative data showing the percentages of non-dividing cells from each culture condition gated by FSC. Data represent mean ^ SEM; ***P < 0.05, two-sided t-test. Data is from six donors in three separate experiments. In these experiments, perforin (D) and granzyme B (E) levels were compared in FSC^high populations of cells expressing control or BCL11B mRNA gated by number of cell divisions. FIG.20 shows a comparison of perforin and granzyme B levels were in FSC^high populations of cells expressing control or BCL11B mRNA gated by number of cell divisions. FIG.21 shows the same analyses as FIG.20 of perforin and granzyme B for the FSC^low populations. Left: Flow cytometry plots of cytotoxic molecule MFIs by cell division from a representative donor. Right: Cumulative data from five donors in three separate experiments. One donor was excluded due to a lack of intracellular staining. Data represent mean ^ SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 2-way ANOVA with multiple comparisons. FIG.22 shows that BCL11B overexpression in iPSC-derived NK cells enhances cytotoxic function. iPSCs engineered with a doxycycline-inducible BCL11B construct were differentiated into CD34⁺ hematopoietic progenitor cells and then differentiated along the NK cell lineage in the presence or absence of doxycycline. After differentiation, iNK cells from both cultures were expanded with engineered feeder cells for seven days and then used for phenotypic analysis and functional testing. (A) Fold increases in iNK cell numbers from cultures with and without doxycycline during iNK cell differentiation and expansion. Data are from three independent differentiations. Data represent mean ^ SEM; *P < 0.05, 2-way ANOVA. (B) Flow cytometry analysis of granzyme B and perforin levels in iNK cells cultured with and without doxycycline in a representative experiment. (C) Cumulative data from three independent differentiation experiments. Data represent mean ^ SEM; *P < 0.05, two-sided t-test. FIG.23 shows data from an experiment wherein iNK cells differentiated with and without doxycycline were used as effectors in 2-D co-culture assays tumor cells labeled with NucLight Red at 4:1 effector-to-target (E:T) ratios. (A) A549 tumor cells; (B) SKOV3 tumor cells. Target cell killing was measured in real time imaging. Results are representative of four independent experiments. Data represent mean ^ SEM; **P < 0.01, ***P < 0.001, 2-way ANOVA of triplicate results. FIG.24 shows results from an experiment wherein iNK cells differentiated with and without doxycycline were used as effectors in co-culture assays with A549 and SKOV3 spheroids at 4:1 E:T ratios. Left: Representative spheroid images at day of the assay. Right: Cumulative data from the spheroid assays. Results are representative of two independent experiments. Data represent mean ^ SEM; ***P < 0.001, ****P < 0.0001, 2-way ANOVA of triplicate results. FIG.25 shows bioluminescence imaging of mice weekly for the duration of IL-2 support in vivo. NSG mice were injected i.p. with 1×10⁵ OVCAR8 ovarian cancer cells expressing luciferase. Groups of mice then received no treatment (tumor alone), 1×10⁷ control iNK cells (no dox), or 1×10⁷ BCL11B iNK cells (dox). Mice receiving iNK cells were also given injections of IL-2 three times per week for three weeks. FIG.26 shows cumulative bioluminescence from day 0 through day 21 of the mice in FIG.25. Data represent mean ^ SD; *P < 0.05, **P < 0.01, 2-way ANOVA with multiple comparisons. Kaplan Meier curves showing probability of survival over time. **P = 0.0068 comparing survival curves of mice receiving control iNK cells to those receiving iBCL11B iNK cells, Log-rank (Mantel-Cox) test FIG.27 shows the frequencies of cell population subsets, as determined by flow cytometry at each time point, after CD56^bright NK cells were transfected with BCL11B or mAzami mRNA and cultured with low-dose IL-15. (A) NKG2A⁺KIR-; (B) NKG2A-KIR⁺; (C) CD16⁺CD57-; (D) CD16⁺CD57⁺. FIG.28 shows the frequencies of cell population subsets after CD56^dimCD57- NK cells were transfected with BCL11B or mAzami mRNA and cultured with low-dose IL-15. (A) NKG2A⁺KIR-; (B) NKG2A⁺KIR⁺. FIG.29 shows the frequencies of cell population subsets, as determined by flow cytometry at each time point, after CD56^dimCD57⁺ NK cells were transfected with BCL11B or mAzami mRNA and cultured with low-dose IL-15. (A) NKG2A⁺KIR-; (B) NKG2A-KIR⁺; (C) CD16⁺CD57-; (D) CD16⁺CD57⁺. FIG.30 shows histogram plots of pSTAT5 levels in the indicated subsets of peripheral blood NK cells during IL-15 stimulation from a representative donor (top) and cumulative data from four donors (bottom). FIG.31 shows mean fluorescence intensity values CD16⁺CD57- and CD16⁺CD57⁺ NK cells gated as FSC^high and FSC^low after bulk CD3-CD56⁺ NK cells were transfected with BCL11B or mAzami mRNA and cultured with low-dose IL-15 for seven days. (A) Granzyme B; (B) perforin. FIG.32 shows T Cell Receptor (TCR)αβ T cells from iBcl11b-iPSCs and functional analysis of iNK cells. (A) The percent of TCRαβ T cells from iBcl11b-iPSC cultured with or without doxycycline. Data represent mean ^ SEM; ***p<0.001. (B) The percent of perforin and granzyme B levels in iNK cells from Bcl11b-KO, No Dox and Dox cultures. Data represent mean ^ SEM; **p<0.001, ***p<0.001. FIG.33 shows that transient induction of Bcl11b during NK cell differentiation enhances natural cytotoxicity against tumor cells. The cytotoxicity of iPSC-derived NK cells differentiated with and without BCL11B induction was determined in real time using the INCUCYTE (Sartorius AG, Göttingen, Germany) imaging system. Shown are the normalized killing curves over the course of 48 hours. DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Hematopoietic cells play numerous important roles in the body. Broadly, hematopoietic cells are classified as lymphoid or myeloid. Natural killer (NK) cells, a type of hematopoietic lymphoid cell, are often used in immunotherapy to treat cancer. For example, NK cells may be differentiated from isolated stem cells and later infused into a patient. NK cells specialize in killing infected or malignant cells and can orchestrate immune responses through the release of chemokines and cytokines. NK cells found within peripheral blood are phenotypically heterogeneous but can be broadly categorized into three distinct groups. CD56^bright NK cells represent a minor subpopulation in blood and are considered immunomodulatory given their ability to rapidly secrete large amounts of inflammatory cytokines and chemokines upon activation. The CD56^bright subset is also highly proliferative and exhibit ten-fold greater proliferation upon exposure to IL-2 relative to cytotoxic CD56^dim NK cells. The maturation of CD56^dim NK cells is marked by acquisition of CD57, and CD57⁺CD56^dim NK cells proliferate significantly less in response to different combinations of cytokines relative to CD57-CD56^dim NK cells. The developmental relationship between CD56^bright and CD56^dim NK cells is unclear. Evidence that CD56^bright NK cells differentiate into CD56^dim NK cells comes from studies where sorted CD56^bright NK cells adopted phenotypic characterizations associated with CD56^dim NK cells after stimulation in vitro. Multiple studies have described NK cell subsets in peripheral blood and bone marrow with intermediate features between CD56^bright and CD56^dim subsets, suggestive of a developmental trajectory. However, evidence that CD56^dim and CD56^bright NK cells are ontologically distinct comes from analyses of NK cells from patients with mutations in the GATA2 gene who lack CD56^bright but not CD56^dim NK cells. Clonal tracking of hematopoiesis in rhesus macaques also demonstrated distinct precursors giving rise to CD16^-/dimCD56⁺ and CD16⁺CD56^-/dim NK cells, which are functional equivalents of CD56^bright and CD56^dim NK cells, respectively, in humans. Differentiating stem cells to derive hematopoietic immune cells, such as NK cells, is technically challenging and often has a relatively low yield. The present disclosure describes cells and methods that may improve the yield of differentiated hematopoietic cells, such as NK cells, that are differentiated from stem cells. Methods of modulating hematopoietic stem cells to produce differentiated cell types are often suboptimal. While generation of engraftable hematopoietic and lympho-myeloid cells from human pluripotent stem cells (hPSCs) is possible, further progress in transitioning these technologies from the laboratory to clinical application is dependent on developing reproducible protocols for producing therapeutic cells reliably under specific physiological conditions. Therefore, to advance the manufacturing of lympho-myeloid cells and hematopoietic stem cells (HSCs), it is imperative to identify the precise molecular pathways and factors governing the specification of multipotent lympho-myeloid progenitors from hPSCs. Broadly, hematopoietic cells differentiate into either lymphoid or myeloid progenitors then commit to a specific identity, such as a T cell or an NK cell. Gene expression in stem cells before or during differentiation directs differentiation of the stem cells. However, the effect of modulating expression of many genes is not well-characterized. The gene BCL11B encodes a protein called B-cell lymphoma/leukemia 11B (BCL11B), a Kruppel-like C2H2-type zinc finger transcription factor required for T cell commitment and development of αβ T cells. BCL11B is involved in immune regulation, and mutations within BCL11B have been implicated in cancer development. As it is used herein, “BCL11B” refers to the protein encoded by the gene “BCL11B.” Expression of BCL11B is dependent upon NOTCH signaling and a cohort of transcription factors. Expression of BCL11B occurs in lymphoid- myeloid clusters of definitive hematopoiesis and hemogenic endothelium from human pluripotent stem cells. However, little is known about the role of BCL11B in human hematopoietic stem/progenitor cell differentiation from iPSCs. The present disclosure reports that BCL11B is a regulator of lympho-myeloid hematopoiesis in iPSCs. Further, the present disclosure reports that increased expression of BCL11B in iPSCs during differentiation increases NK cell-mediated cytotoxicity. Introduction of BCL11B mRNA considerably slowed NK cell proliferation while simultaneously driving maturation. Induced pluripotent stem cell (iPSC)-derived NK cells (iNK cells) with inducible BCL11B expression exhibited faster and more complete solid tumor cell killing in vitro and significantly better tumor control in vivo. In addition, inducible expression of BCL11B in iPSCs during differentiation resulted in increased TCRαβ T cells. These findings suggest that increasing BCL11B expression in hematopoietic cells, such as T cells and NK cells, may improve their function and production from iPSCs. As is described herein, BCL11B markedly slowed NK cell proliferation in response to IL-15 and led to the upregulation of cyclin-dependent kinase inhibitors. Slower rates of proliferation were associated with increases in NK cell granularity and cytotoxic molecule levels. Independent of its regulation of NK cell proliferation, BCL11B promoted acquisition of CD57 within the CD56^dim subset. To test the impact of BCL11B overexpression on the antitumor activity of NK cells, induced pluripotent stem cell (iPSC)-derived NK (iNK) cells were generated with inducible BCL11B expression. iNK cells with BCL11B overexpression mediated superior antitumor activity both in vitro and in a xenogeneic adoptive transfer model of ovarian cancer. While NK cells have distinct innate properties, they also share characteristics associated with adaptive CD8⁺ T cells, including cytotoxicity and a conserved program underlying memory. BCL11B is important for CD8⁺ T cell cytotoxic function, expansion, and memory formation in mice. A gradient of BCL11B expression exists throughout NK cell differentiation with very low levels in CD16-CD56^bright NK cells and upregulation in the small fraction of cells with a CD16^intCD56^bright phenotype. BCL11B levels increase markedly in less mature NKG2A⁺KIR- CD57-CD56^dim NK cells and continue to increase incrementally throughout canonical NK cell maturation, with the highest levels observed in cytomegalovirus (CMV)-induced adaptive NK cells. These findings in human NK cells somewhat diverge from those reported in mice, where early NK cell development is normal in Bcl11b-deficient mice, and Bcl11b is transiently expressed in immature NK cells but not in NK cell precursors or mature NK cells. Nonetheless, CMV-induced adaptive NK cell expansions were compromised in Bcl11b-deficient mice. Thus, there are important regulatory differences between humans and mice with respect to NK cell development. This is supported by comparisons of regulatory regions between human and mouse NK cells, where high conservation between human CD56^bright and murine CD27⁺CD11b- cells but far less overlap between human CD56^dim and murine CD27⁺CD11b- cells was observed. Such differences could be a consequence of the pathogen-free environment of laboratory mice, or they may reflect fundamental differences in the transcription factor circuitry that control NK cell differentiation between species. Nucleic acids As used herein, the term “nucleic acid” or “oligonucleotide” refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include but are not limited to genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules such as aptamers, plasmids, anti- sense DNA strands, shRNA, ribozymes, nucleic acids conjugates, and oligonucleotides. A nucleic acid may be single-stranded, double-stranded, linear, or covalently circularly closed molecule. A nucleic acid can be isolated. The term “isolated nucleic acid” means that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, (iv) was synthesized, for example, by chemical synthesis, or (vi) extracted from a sample. A nucleic might be introduced—i.e., transfected—into cells. When RNA is used to transfect cells, the RNA may be modified by stabilizing modifications, capping, or polyadenylation. In one aspect, the present disclosure describes a nucleic acid including a sequence of the gene BCL11B. In one or more embodiments, the nucleic acid includes deoxyribonucleic acid (DNA), such as double-stranded DNA (dsDNA). Additionally, or alternatively, the nucleic acid may include ribonucleic acid (RNA), and it may be single-stranded. Examples of suitable forms of nucleic acids include plasmid DNA, mRNA, dsDNA, ssDNA, and genomic DNA. In one or more embodiments, the nucleic acid includes mRNA. The mRNA may be reverse transcribed in vitro. An mRNA reverse transcribed in vitro may be administered to a cell or to a subject. In one or more embodiments, the mRNA may include elements to enable detection, such as a fluorescent marker. In one or more embodiments, the mRNA may include the BCL11B open reading frame (ORF). In contrast to other species of nucleic acids, mRNA advantageously allows for transient expression of a gene with decreased likelihood of long-term expression. In this way, expression of the gene may be temporally controlled by administering the mRNA only when expression of the gene is desired. Methods of reverse transcribing mRNA in vitro are known to the art and include use of a T7 RNA polymerase and corresponding T7 RNA promoter. In one or more embodiments, the mRNA is transcribed from a precursor DNA, such as a plasmid. The plasmid may include elements for mRNA expression, such as a promoter, a terminator, and a polyadenylation sequence. A nucleic acid may be introduced into a cell using any known method. For example, an mRNA may be introduced into a cell using transfection, electroporation, or lipofection. In some embodiments, a nucleic acid is introduced into a cell using a vector, such as a viral vector or a lipid nanoparticle. In one or more embodiments, the nucleic acid is isolated. Generally, a nucleic acid can be extracted, isolated, amplified, or analyzed by a variety of techniques well known to those of ordinary skill in the art. Examples of nucleic acid analysis include, but are not limited to, sequencing and DNA-protein interaction. Sequencing may be by any method known in the art. DNA sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, and next generation sequencing methods such as sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina/Solexa sequencing, allele specific hybridization to a library of labeled oligonucleotide probes, sequencing by synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, and SOLiD sequencing. Separated molecules may be sequenced by sequential or single extension reactions using polymerases or ligases as well as by single or sequential differential hybridizations with libraries of probes. In one or more embodiments, the nucleic acid includes a promoter. Typically, the promoter regulates expression of BCL11B and/or one or more other proteins encoded by the expression cassette. The promoter may be an inducible promoter, such as the TET promoter. In one or more embodiments, the nucleic acid includes one or more sequences that encode a reporter protein. A reporter protein may include a fluorescent protein or a detectable label. In one or more embodiments, the nucleic acid includes one or more sequences that encode a selectable marker. A selectable marker may confer resistance to a condition, such as being cultured with an antibiotic. In another aspect, this disclosure describes an isolated nucleic acid sequence that encodes any embodiment of BCL11B or any component fragment of BCL11B, having the amino acid sequence of any one of human BCL11B (UniProt No. Q9C0K0) or murine BCL11B (Q99PV8). In one or more embodiments, the isolated nucleic acid encodes human BCL11B (UniProt No. Q9C0K0) or murine BCL11B (Q99PV8). Given the amino acid sequence of any BCL11B, or one or more component fragments of the BCL11B, a person of ordinary skill in the art can determine the full scope of polynucleotides that encode that amino acid sequence using conventional, routine methods. In one or more embodiments, the nucleic acid includes the full coding sequence of the human BCL11B gene (NCBI Gene ID: 64919). In one or more embodiments, the nucleic acid includes the coding sequence (CDS) of the human BCL11B gene. The CDS of the human BCL11B gene may include the CDS for any isoform, such as isoform 1, 2, 3, 4, X1, X2, X3, or X4. In one or more embodiments, the nucleic acid includes the CDS of isoform 1 of the human BCL11B gene. In another aspect, the present disclosure relates to a vector including a nucleic acid encoding the gene BCL11B. As it is used herein, a vector is a compound capable of transferring a nucleic acid to a cell. In one or more embodiments, the vector is a viral vector. Examples of viral vectors include lentiviral, retroviral, adenoviral, rabies, herpes, and adeno-associated viral vectors. Some viral vectors are capable of delivering a nucleic acid to a cell and integrating the nucleic acid into the cell’s genome. In one or more embodiments, the vector is a non-viral gene delivery vector. Examples of non-viral vectors include lipid nanoparticles, gold nanoparticles, liposomes, and synthetically derived nanoparticles. A vector described herein may be used to deliver a nucleic acid to a cell, either in culture or in an organism. Engineered cells In one aspect, the present disclosure describes an engineered cell including a nucleic acid encoding the gene BCL11B. Typically, the nucleic acid encoding the gene BCL11B is an exogenous nucleic acid, meaning that it does not naturally exist within the cell without intervention. The nucleic acid encoding BCL11B is typically consistent with nucleic acids encoding BCL11B described herein. In one or more embodiments, the gene BCL11B is stably integrated into the genome of the cell. For example, the gene BCL11B may be integrated at a safe harbor locus, such as the AAVS1 locus. Alternatively, the engineered cell may include a stably maintained nucleic acid encoding the gene BCL11B that is not integrated into the genome, such as a viral episome. In one or more embodiments, the gene BCL11B is stably integrated into the genome of the cell in the context of a transposon or a recombinase. For example, the gene may be flanked by loxP sites or inverted terminal repeats (ITRs). In one or more embodiments, the cell is a stem cell. The stem cell may be a wild-type stem cell or it may be a manipulated stem cell, such as an induced pluripotent stem cell (iPSC). In one or more embodiments, the iPSC is a CD34⁺ cell. In one or more embodiments, the cell is a common myeloid progenitor cell. In some such embodiments, the cell may be differentiated to form a myeloid cell, such as a neutrophil, monocyte, macrophage, dendritic cell, megakaryocyte, eosinophil, or basophil. In one or more embodiments, the cell is a common lymphoid progenitor cell. In some such embodiments, the cell may be differentiated to form a lymphoid cell, such as a T cell, B cell, or NK cell. In one or more embodiments, the cell is a T cell, B cell, or NK cell. In one or more embodiments, the NK cell is a CD56^bright NK cell. In one or more embodiments, the NK cell is a CD56^dim NK cell. In one or more embodiments, the NK cell is CD57⁺. In one or more embodiments, the NK cell is CD16⁺. In one or more embodiments, the cell is a CD8⁺ T cell. In one or more embodiments, the cell is a CD4⁺ T cell. As is described herein, Bcl11b knockout CD4⁺ T cells exhibit enhanced proliferation and viability when activated for several days in vitro. In one or more embodiments, the cell is a TCRαβ T cell. Methods of modulating cells In one aspect the present disclosure relates to a method of modulating a stem cell including increasing expression of BCL11B and differentiating the stem cell. Methods of differentiating stem cells are known to the art, and most methods of differentiation are thought to be compatible with increased expression of BCL11B. In one or more embodiments, increasing expression of BCL11B includes inducing expression of BCL11B. For example, if a cell includes an inducible expression cassette encoding BCL11B, increasing expression of BCL11B may include treating the cell to activate the inducible promoter. Notably, inducible expression of a gene, such as BCL11B, may allow a user to turn expression of the gene on and off at predetermined points in time. In this way, it is possible to regulate expression of a gene, such as BCL11B, such that it is expressed during one or more particular periods of time. In one or more embodiments, a method includes inducing expression of BCL11B and subsequently stopping induction, thereby decreasing or stopping expression of BCL11B. In one or more embodiments, the method includes increasing expression of BCL11B during differentiation of the stem cell, such as hematopoietic differentiation. In one or more embodiments, the method includes increasing expression of BCL11B during the mesoderm stage of hematopoietic differentiation. Typically, the mesoderm stage occurs 2-4 days after hematopoietic differentiation has begun. Additionally or alternatively, the method may include increasing expression of BCL11B during differentiation of one or more terminally differentiated myeloid or lymphoid cells. In one or more embodiments, the method includes increasing expression of BCL11B during NK cell differentiation. In one or more embodiments, the method includes increasing expression of BCL11B during T cell differentiation. As is described herein, increasing expression of BCL11B during both early hematopoietic differentiation and terminal maturation may advantageously increase activation of the differentiated cell. In one or more embodiments, the cell used in the method is a stem cell. The method may be compatible with isolated wild-type stem cells or induced pluripotent stem cells (iPSCs). The method may be compatible with hematopoietic stem cells (HSCs). In one or more embodiments, the stem cell is CD34⁺. In one or more embodiments, inducing expression of BCL11B includes adding a molecule to induce expression of an inducible promoter. Inducible promoters are described herein and include, for example, the TET-ON system, which uses doxycycline to induce gene expression. In one or more embodiments, inducing expression of BCL11B includes adding a molecule to induce expression to the stem cell at a concentration of at least 0.1 µg/mL, at least 0.2 µg/mL, at least 0.3 µg/mL, at least 0.4 µg/mL, at least 0.5 µg/mL, at least 0.6 µg/mL, at least 0.7 µg/mL, at least 0.8 µg/mL, at least 0.9 µg/mL, at least 1 µg/mL, at least 1.5 µg/mL, at least 2 µg/mL, at least 2.5 µg/mL, at least 3 µg/mL, at least 4 µg/mL, or at least 5 µg/mL. In one or more embodiments, the method increases the number of differentiated cells committed to lymphoid lineage compared to a method wherein expression of BCL11B is not induced. Lymphoid cells are described in greater detail herein and include T cells, B cells, and NK cells. In one or more embodiments, the cell is a T cell, B cell, or NK cell. In one or more embodiments, the cell is a CD8⁺ T cell. In one or more embodiments, the method further includes isolating a differentiated cell. In one or more embodiments, the method increases formation of arterial hemogenic endothelial (AHE) cells formed from a treated stem cell compared to a stem cell wherein expression of BCL11B is not induced. AHE cells are progenitor cells that will eventually form the lining of blood vessels. Typically, AHE cells are defined as cells that are KDR⁺CD144⁺CD34⁺CXCR4⁺. The number of AHE cells present in a culture may be determined using any suitable method including flow cytometry or fluorescence-activated cell sorting (FACS). In one or more embodiments, the method increases formation of multipotent hematopoietic progenitors (MHPs) formed from a treated stem cell compared to a stem cell wherein expression of BCL11B is not induced. MHPs, also referred to as “hemocytoblasts” are stem cells that have committed to a hematopoietic lineage but may still form a myeloid or a lymphoid cell. Typically, MHPs are defined as cells that are CD34⁺CD43⁺CD45⁺. The number of MHPs present in a culture may be determined using any suitable method including flow cytometry or FACS. In one or more embodiments, the method results in NK cells with enhanced cytotoxicity compared to NK cells prepared from stem cells in which expression of BCL11B was not induced. The level of cytotoxicity of an NK cell may be measured by coculturing the NK cell with target cells and measuring the rate at which the NK cell kills the target cells. Other methods of characterizing NK cell cytotoxicity include short-term co-culture of NK cells with tumor cells that are labeled with fluorescent dyes or radioactive chromium. In one or more embodiments, the method includes transfecting or transforming the cell to introduce a nucleic acid encoding BCL11B. The cell may be transiently transformed to include the nucleic acid. Alternately, the cell may be stably transformed to include the nucleic acid. Transformation of the cell may be stable or transient. The term “transient transformation” or “transiently transformed” refers to the introduction of one or more transgenes into a cell in the absence of integration of the transgene into the host cell’s genome. Transient transformation may be detected by, for example, enzyme-linked immunosorbent assay (ELISA) that detects the presence of a polypeptide encoded by one or more of the transgenes. Alternatively, transient transformation may be detected by detecting the activity of the protein encoded by the transgene. The term “transient transformant” refers to a cell that has transiently incorporated one or more transgenes. In contrast, the term “stable transformation” or “stably transformed” refers to the introduction and integration of one or more transgenes into the genome of a cell. The term “stable transformant” refers to a cell that has stably integrated one or more transgenes into the genomic DNA. Thus, a stable transformant is distinguished from a transient transformant in that, whereas genomic DNA from the stable transformant contains one or more transgenes, genomic DNA from the transient transformant does not contain a transgene. Methods for both transient and stable expression of coding regions are well known in the art. In one or more embodiments, the method includes stably integrating a nucleic acid encoding BCL11B into the cell. Methods of stably integrating nucleic acids into cells are known to the art and include transposons and gene editing methods that utilize homology-directed repair, such as CRISPR/Cas9. In the preceding description and following claims, the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended—i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). As used herein, “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” or the like are used in their open-ended inclusive sense, and generally mean “include, but not limited to,” “includes, but not limited to,” or “including, but not limited to.” Further, wherever embodiments are described herein with the language “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” and the like, otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided. The term “consisting of” means including, and limited to, that which follows the phrase “consisting of.” That is, “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. The term “consisting essentially of” indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. As used herein, the word “exemplary” means to serve as an illustrative example and should not be construed as preferred or advantageous over other embodiments. As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention. In the preceding description, particular embodiments may be described in isolation for clarity. Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” “one or more embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment except where the features are necessarily mutually exclusive. In several places throughout the above description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order. And, as appropriate, any combination of two or more steps may be performed simultaneously. EXEMPLARY EMBODIMENTS Embodiment 1. A method of modulating hematopoietic differentiation in a stem cell, the method comprising inducing expression of BCL11B in the stem cell and differentiating the stem cell to form a differentiated cell. Embodiment 2. The method of Embodiment 1, wherein the differentiated cell comprises a lymphoid cell. Embodiment 3. The method of Embodiment 2, wherein the lymphoid cell is an NK cell. Embodiment 4. The method of Embodiment 3, wherein the NK cell is a CD56^bright NK cell. Embodiment 5. The method of Embodiment 2, wherein the lymphoid cell is a T cell. Embodiment 6. The method of Embodiment 5, wherein the T cell is a CD8⁺ T cell. Embodiment 7. The method of any preceding Embodiment, wherein expression of BCL11B is induced during the mesoderm stage of hematopoietic differentiation. Embodiment 8. The method of any preceding Embodiment, wherein expression of BCL11B is induced during NK cell differentiation. Embodiment 9. The method of any preceding Embodiment, wherein inducing expression of BCL11B comprises inducing expression and subsequently stopping induction. Embodiment 10. The method of any preceding Embodiment, wherein the method increases formation of arterial hemogenic endothelial (AHE) cells compared to a stem cell in which expression of BCL11B is not induced. Embodiment 11. The method of any preceding Embodiment, wherein the method increases formation of multipotent hematopoietic progenitor (MHP) cells compared to a stem cell in which expression of BCL11B is not induced. Embodiment 12. The method of any preceding Embodiment, wherein the method increases NK cell cytotoxicity compared to an NK cell differentiated from stem cells in which expression of BCL11B is not induced. Embodiment 13. The method of any preceding Embodiment, wherein the stem cell is an induced pluripotent stem cell (iPSC) or a hematopoietic stem cell (HSC). Embodiment 14. The method of any preceding Embodiment, wherein BCL11B expression is induced in the stem cell in vitro. Embodiment 15. The method of any preceding Embodiment, further comprising isolating the differentiated cell. Embodiment 16. A nucleic acid including a sequence of BCL11B and an inducible promoter, wherein expression of BCL11B is controlled by the inducible promoter. Embodiment 17. The nucleic acid of Embodiment 16, further comprising a selectable marker. Embodiment 18. A gene expression vector comprising the nucleic acid of Embodiment 16 or Embodiment 17. Embodiment 19. An mRNA produced by the nucleic acid of Embodiment 16, wherein the mRNA comprises the open reading frame of BCL11B. Embodiment 20. An engineered cell comprising the nucleic acid of any preceding Embodiment. Embodiment 21. The engineered cell of Embodiment 20, wherein the cell is a stem cell, such as an iPSC or an HSC. Embodiment 22. The engineered cell of Embodiment 20 or Embodiment 21, wherein the nucleic acid is integrated into the genome of the cell. EXAMPLES The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein. Example 1 Generation of iBCL11B-iPSC cell line In this Example, BM-iPSCs (BM9) were genetically engineered to harbor a cassette for inducible expression of BCL11B. Cell culture BM-iPSCs (BM9) from WiCell were cultured on Matrigel-coated plate (BD Biosciences, Franklin Lakes, NJ) in mTeSRplus media (STEMCELL Technologies Inc., Vancouver, Canada) with daily media changes. Cells were split weekly by dissociation with 0.5 mM EDTA (Sigma- Aldrich, St. Louis, MO). iPSC cultures were visualized daily by phase contrast microscopy. The components of media used during iPSC differentiation is described in Table 1. Table 1. Media Compositions. Component B0 Media iNK Media-A iNK Media-B iNK Media-C

Pen/Strep 5 mL 5 mL 5 mL 5 mL 2-ME 20 μM 20 μM 20 μM 20 μM

Constructon o vectors and generat on o C - SC ne To target the donor cassette to the AAVS1 locus in BM-iPSC, a human BCL11B gene, a CAG promoter, and the fluorescent protein VENUS were cloned into an AAVS1-SA-2A-Puro vector. CRISPR/Cas9 nuclease system was used to target at the AAVS1 locus (FIG.1).2×10⁶ single cells dissociated from BM-iPSCs were resuspended in NUCLEOFECTOR solution (Lonza, Basel, Switzerland) and electroporated with 1 μg of Cas9 protein (PNA Bio, Inc., Thousand Oaks, CA), 1 μg of sgRNA and 1 μg of AAVS1-SA-2A-Puro-BCL11B-VENUS donor plasmid. Cells were subsequently plated at a low density on MATRIGEL (Discovery Labware, Inc., Bedford, MA)-coated six-well plate in mTeSR1plus medium supplemented with CloneR2 (STEMCELL Technologies Inc., Vancouver, Canada). The culture was maintained for five days, following which 1 μg/mL puromycin was added. Cells were cultured for up to ten days in puromycin until colonies formed. After seven to ten days, ten puromycin-resistant colonies were picked and expanded. Genotyping of iBCL11B-iPSC clones Genomic DNA of puromycin-resistant colonies was extracted using a commercial kit (ZR-96 Quick-gDNA kit, Zymo Research Corp., Irvine, CA). For positive genotyping, the following primer pair was used: 5′-CTGTTTCCCCTTCCCAGGCAGGTCC-3′ (SEQ ID NO:1) and 5′-TCGTCGCGGGTGGCGAGGCGCACCG-3′ (SEQ ID NO:2, Tm= 65 °C). For homozygous genotyping, the following set of primer sequences was used: 5′- CGGTTAATGTGGCTCTGGTT-3′ (SEQ ID NO:3) and 5′-GAGAGAGATGGCTCCAGGAA- 3′ (SEQ ID NO:4, T_m = 60 °C). RNA was extracted from each clone cultured with or without doxycycline and used to prepare cDNA. RT-qPCR was used to amplify a BCL11B transcript. A gel showing PCR products from the ten puromycin-resistant clones is shown in FIG.2A. All clones except clone 2 were found to produce BCL11B transcript when treated with doxycycline, indicating that they harbored the BCL11B expression cassette. Karyotypes of iPSCs treated to incorporate the BCL11B expression cassette and untreated iPSCs were prepared to check for large-scale genetic abnormalities. No chromosomal rearrangements were observed in treated iPSCs, indicating that incorporation of the BCL11B expression cassette did not induce any large-scale genetic abnormalities (FIG.2B). Clone 10p was selected for use in subsequent examples. Functional characterization of iBCL11B-iPSC clones iBCL11B-iPSCs were cultured in mTeSRplus (STEMCELL Technologies Inc., Vancouver, Canada).24 hours after plating the cells, 2 µg/mL of doxycycline was added to the culture media. Cells were harvested and analyzed using flow cytometry at 0, 3, 6, 12, 24, 48, 72, 96, and 120 hours after doxycycline was added. Cells were gently pipetted and filtered through a 70 µm or 100 μm strainer sitting on a 50 mL tube. The cells were then pelleted by centrifugation and washed twice with PBS -/- solution containing 1% bovine serum albumin (BSA). The cells were stained with appropriate conjugated antibodies for 25 minutes at room temperature in the dark, and analyzed via flow cytometry after washing with BSA containing solution. FLOWJO software (Becton Dickenson & Co., Franklin Lakes, NJ) was used to process the flow data. VENUS expression was used as a proxy measurement of BCL11B expression. The highest expression of VENUS was observed 48 hours following doxycycline addition, with expression appreciably increasing 6 hours and decreasing 72 hours following doxycycline addition (FIG.3). In addition, cells were cultured for 24 hours with a range of doxycycline concentrations from 0.5 µg/mL to 6 µg/mL. Cells were harvested and analyzed for expression of VENUS using flow cytometry. The highest expression of VENUS was observed in cells cultured with 5 µg/mL of doxycycline (FIG.4). However, greater than 70% of cells expressed VENUS when treated with at least 1 µg/mL of doxycycline (FIG.4). Example 2 Generating arterial hemogenic endothelium by inducing BCL11B expression at the mesoderm stage Hematopoietic differentiation of hPSCs iBCL11B-iPSCs were differentiated into hematopoietic lineages using a 3D-organoid- based differentiation protocol. Briefly, on day one, hPSCs were singularized and resuspended in an organoid medium.30-40 μL droplets were deposited on square plates such that each droplet contained about 2500-3500 cells. The plate was closed, inverted, and incubated overnight to facilitate hanging drop cell aggregation. The following day, the droplets were harvested with phosphate-buffered saline or iPSC medium, centrifuged, and resuspended in differentiation medium supplemented with BMP4, VEGF, FGF (25 µg/mL to 50 µg/mL), Activin A (5 µg/mL to 15 µg/mL) and ROCK inhibitor (1 µM to 10 µM) and plated on collagen-IV coated plates and cultured in hypoxic conditions (5% O₂, 5% CO₂). On day two, the media was changed to differentiation medium supplemented with VEGF (25 ng/mL to 50 ng/mL), FGF (25 ng/mL to 50 ng/mL), BMP4 (1 ng/mL to 10 ng/mL), CHIR99021 (1 µM to 3 µM), SB-431542 (2 µM to 5 µM) and returned to incubate in hypoxic conditions. On day three, the medium was replaced with differentiation medium supplemented with VEGF (25 ng/mL to 50 ng/mL), FGF (25 ng/mL to 50 ng/mL), BMP4 (1 ng/mL to 10 ng/mL) and placed back in the hypoxia incubator. On day five, the medium was replaced with differentiating medium supplemented with SCF(25 ng/mL to 50 ng/mL), VEGF (25 ng/mL to 50 ng/mL), TPO (25 ng/mL to 50 ng/mL) IL-6 (25 ng/mL to 50 ng/mL), FGF (5 ng/mL to 15ng/mL), IL-3 (1 ng/mL to 10 ng/mL) and the plates were incubated in normoxic conditions (5% CO2). On day five, cells were sorted by fluorescence activated cell sorting (FACS) to isolate hemogenic endothelium subsets for further studies. On day seven, additional differentiation medium supplemented SCF, VEGF, TPO, IL-6 (25 ng/mL to 50 ng/mL), FGF, IL-3 (1 ng/mL to 10 ng/mL) was added. A first group of cells was treated with doxycycline at a concentration of Doxycycline was added to the cells at a concentration of 2 ^g/mL on day two of differentiation. Cells were collected on day five and analyzed for formation of hemogenic endothelium cells using flow cytometry. Cells derived from iBCL11B-iPSC treated with doxycycline and iBCL11B-iPSC not treated with doxycycline were analyzed. When cells were treated with doxycycline, the number of arterial hemogenic endothelium (KDR⁺CD144⁺CXCR4⁺) cells increased significantly (FIG.5). These results indicate that expression of BCL11B during mesoderm stage increased the formation of arterial hemogenic endothelial cells. Cells were collected on day nine or day 10 and analyzed using flow cytometry. Cells derived from iBCL11B-iPSCs treated with doxycycline and iBCL11B-iPSC not treated with doxycycline were analyzed. When cells were treated with doxycycline, the number of multipotent hematopoietic progenitors (CD34⁺CD43⁺CD45⁺) cells increased significantly (FIG. 6). These results indicate that expression of BCL11B during mesoderm stage increased the formation of multipotent hematopoietic progenitor cells. Cells were collected on day 30 and analyzed for expression of surface receptors known to regulate NK cell effector function. Cells derived from iBCL11B-iPSCs treated with doxycycline were found to express higher levels of NKG2A, NKp30, NKp44, CNAM1, and CD117 (FIG.7), suggesting that overexpression of BCL11B also improves in vitro NK cell differentiation from iPSCs. This Example demonstrated the stage-specific roles of BCL11B overexpression in enhancing NK cell differentiation from iPSCs. Example 3 Characterization of the effect of BCL11B expression on NK cell effector function OP9-DLL4 Culture and Irradiation OP9-DLL4 cells were used for NK-lymphoid differentiation. OP9-DLL4 cells are maintained in aMEM (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA) containing 20% HYCLONE (Cytiva, Marlborough, MA) fetal bovine serum (FBS) and analyzed using flow cytometry at every passage, post-irradiation and post-cryopreservation to monitor the integrity of the cells. For irradiation, cells were harvested using a cell detachment solution (ACCUTASE, Innovative Cell Technologies, San Diego, CA) and centrifuged at 300×g for five minutes. Cells were resuspended and combine pellet cells volumes into one 50 mL conical tube with 40 mL OP9-DLL4 media. The top of tube was wrapped with parafilm and placed tube on ice and taken for irradiation. The cells were irradiated at 5000 rads and returned to ice after irradiation. Irradiated cells were centrifuged at 300×g for five minutes and cryopreserved at a density of 2×10⁶ cells/vial for NK differentiation. NK-lymphoid differentiation The irradiated OP9-DLL4 stromal cells were thawed and plated at a density of 4.47×10⁴ cells/cm² in aMEM (INVITROGEN, Thermo Fisher Scientific, Inc., Waltham, MA). The cells were cultured overnight at 37 °C, 5% CO₂ to allow cells to attach. OP9 cells were at least 90% confluent before seeding iCD34. CD34⁺ enriched MHP cells (1.5×10⁴ cells/cm²) were resuspended in appropriate volume of iNK media-A, described in Table 1. Three days after plating, cells were supplemented with 50% of initial plating volume iNK media-B with or without 2 µg/mL doxycycline. On day 7, cells were supplemented with 50% of initial plating volume iNK media-C with or without 2 µg/mL doxycycline. On day 10, cells were harvested by pipetting up and down to dislodge iNK cells and any remaining OP9-DLL4 cells. Cells were strained using a 40 µm filter to achieve a single cell suspension, centrifuged, and resuspended into iNK media-C with or without 2 µg/mL doxycycline for further differentiation without feeder OP9-DLL4 stromal cells. Cells were counted and seeded into a tissue culture plate at a density of 2.5×10⁵ cells per cm² in iNK media-C with or without doxycycline. Cells were cultured for another 10 days. On days 13 and 17, 50% of the initial plating volume of iNK media-C with or without 2 µg/mL doxycycline was added. At day 20, a portion of the cells were harvested, counted, centrifuged, and phenotypically analyzed using a flow cytometer. The remaining cells were expanded using irradiated K562 cells in media without doxycycline was added to the cells on days 23 and 27. Cells were harvested and analyzed using flow cytometry on day 27. NK cell expansion Differentiated NK cells derived from iBCL1B-iPSCs treated with doxycycline or not treated with doxycycline were counted and expanded using irradiated K562/mbIL21/41BBL feeder cells. NK cells were resuspended in B0 medium supplemented with IL-2 (100-500 U/mL) and seeded at 2.5×10⁵ cells/cm². Irradiated feeder cells were combined with CD56⁺ NK cells at a ratio of 2:1. Media supplemented with IL-2 was added to the culture once every two days. NK cell-mediated in vitro cytotoxicity assay SKOV3 cells transduced with NUCLIGHT Red (Sartorius AG, Göttingen, Germany) were seeded into 96-well plates (Corning Life Sciences, Corning, NY). The target cell survival was assessed in real-time using an INCUCYTE Live Cell Analysis System (Essen BioScience, Ann Arbor, MI). Live cell numbers were quantified with INCUCYTE S3 software (Essen BioScience Ann Arbor, MI) and normalized to the number of live cells remaining in the target cell-only control group. SKOV3 cells were cocultured with NK cells at a ratio of either 2 or 4 NK cells per SKOV3 cells. Cultures of SKOV3 with NK cells derived from iBCL11B-iPSC cells treated with doxycycline during NK cell differentiation and NK cells derived from untreated iBCL11B-iPSC cells were prepared at each ratio. The percentage of live SKOV3 cells in each culture was measured hourly for 48 hours (FIG.33). Particularly when a ratio of 2:1 NK cells to SKOV3 cells was used, treatment with doxycycline was shown to increase NK cell cytotoxicity. These results indicate that NK cells derived from iPSCs expressing BCL11B exhibit increased cytotoxicity. To determine the effect of BCL11B expression at different timepoints during NK cell differentiation, NK cells were prepared from iBCL11B-iPSC cells treated with 2 µg/mL doxycycline during the mesodermal stage of hematopoietic differentiation (Dox), during NK cell differentiation (No Dox+Dox), or both (Dox+Dox). SKOV3 ovarian tumor cells transduced with NucLight Red were plated in triplicate and allowed to adhere overnight. Each group of NK cells was subsequently cocultured with the SKOV3 cells at a ratio of 2:1 NK cells to SKOV3 cells, and cytotoxicity was measured. Cells treated with doxycycline during NK cell differentiation only exhibited the lowest level of cytotoxicity against SKOV3 cells (FIG.33). Cells treated with doxycycline during the mesodermal stage of hematopoietic differentiation only exhibited increased levels of cytotoxicity (FIG.33). Cells treated with doxycycline exhibited the highest levels of cytotoxicity (FIG.33). Example 4 In-house generation of a modified BCL11B mRNA for primary human NK cell transfection The relative resistance of NK cells to viral transduction and plasmid-based transfection has been a significant barrier to the successful execution of mechanistic studies. Transduction efficiency has improved with the use of baboon envelope pseudotyped lentivectors, but rates remain low in the absence of robust ex vivo expansion. To overcome this barrier, a synthetic mRNA was designed having a CleanCap AG compatible T7 RNA promoter for transcript expression and mRNA stability (Henderson et al., 2021. Curr. Protoc.1:e39), the 5´ UTR of the human beta-globin gene, the BCL11B open reading frame (ORF) modified to reduce uridine content with a T2A cleavage site and an mAzami reporter, a 3´ AES_mtRNR1 UTR for enhanced stability and expression (Orlandini von Niessen et al., Mol. Ther.27:824-836), and a segmented poly(A) tail for reduced recombination (Trepotec et al., 2019. RNA.25:507-518). A control mRNA lacking the BCL11B ORF was created as a control (FIG.9A). To prepare synthetic mRNA, the BCL11B and mAzami control mRNAs were created in two cloning steps using the Mammalian ToolKit (MTK) based on the Golden Gate method. Briefly, a plasmid functionally equivalent to MTK0_027 from the MTK system was generated by amplifying the backbone of MTK2_023 with Q5 High-Fidelity 2× Master Mix (M0492; New United Kingdom Biolabs, Inc., Ipswich, MA). The residual plasmid was digested with DPN1 (R0176; New United Kingdom Biolabs, Inc., Ipswich, MA) and then assembled with the HiFi DNA Assembly Cloning Kit (E5520; New United Kingdom Biolabs, Inc., Ipswich, MA) in combination with the MTK0_027 assembled eblock to create MTK0_027_assembled. This was then used, in combination with additional eblocks, the final vector plasmids. The BCL11B sequence was codon optimized for human expression and then uridine depleted using the Benchling codon optimization tool. The BCL11B sequence was then separated into four eblock fragments and cloned using Golden Gate assembly. Final sequences were confirmed using either Sanger sequencing or whole plasmid sequencing (GENEWIZ, Azenta Life Sciences, South Plainfield, NJ). To express mRNA in vitro, 25 µg of plasmid DNA was digested using 20 µl Sap1 (R0569; New United Kingdom Biolabs, Inc., Ipswich, MA) in a 500 ^1 reaction for two hours at 37 ºC and then inactivated for 20 minutes at 65 °C. A DNA cleanup was then performed using the Bomb.bio #4.2 cleanup carboxyl protocol with Sera-Mag SpeedBeads (09-981-121; Cytiva, Marlborough, MA). mRNA was produced as described previously using N1- Methylpseudouridine-5´-Triphosphate (N-1081-5; TriLink BioTechnologies, San Diego, CA) and CleanCap Reagent AG (3´ OMe) (N-7413; TriLink BioTechnologies, San Diego, CA). To assess RNA integrity and possible degradation, BCL11B and control mRNAs were visualized by gel electrophoresis. Both mRNAs migrated at their expected sizes (1235 bp for the mAzami mRNA and 3896 bp for the BCL11B mRNA) with distinct bands indicative of high RNA quality (FIG.9B). Next, the mAzami control and BCL11B mRNAs were transfected into enriched peripheral blood NK cells stimulated overnight with 10 ng/ml IL-15. mAzami reporter expression levels were analyzed after 24 hours of culture with 1 ng/ml IL-15 by flow cytometry. Both mRNAs were expressed at high frequencies with an average of 77% for the mAzami control mRNA and 64% for the BCL11B mRNA (FIG 10). qPCR was performed to determine expression of the BCL11B transcript. Total RNA was purified from NK cells using the RNeasy Mini Kit (74104; Qiagen, Hilden, Germany), and DNA was digested using DNAse I (18068-015; Invitrogen, Waltham, MA) according to the manufacturer’s instructions. Complementary DNA was synthesized from RNA using a high- capacity RNA-to-cDNA kit (4387406; Applied Biosystems, Foster City, CA), and thermocycle reactions were performed using the Power SYBR Green PCR Master Mix (4367659; Applied Biosystems, Foster City, CA). Primers used included SEQ ID NOs:5-8. High levels of BCL11B transcript in transfected NK cells was confirmed by quantitative RT-PCR (FIG.11A). To ensure that BCL11B protein was present in transfected cells, Western blots were performed using NK cells from two donors 48 hours after transfection with the mAzami control and BCL11B mRNAs. To prepare Western blots, cells were lysed with RIPA Lysis and Extraction Buffer (89900; Thermo Fisher Scientific, Inc., Waltham, MA) supplemented with a protease/phosphatase inhibitor cocktail (5872; Cell Signaling Technology, Inc., Danvers, MA). Proteins were run on an SDS 4-14% Bis-Tris gel and then transferred to a nitrocellulose membrane using the iBlot2 Dry Blotting System (IB21001; Invitrogen, Waltham, MA). Blocking, washing, and antibody incubations were performed with the iBind Flex device (SLF2000; Invitrogen, Waltham, MA). Primary antibodies: rabbit anti-beta-actin (4790; Cell Signaling Technology, Inc., Danvers, MA) and rat anti-BCL11B (ab18465; Abcam, Cambridge, United Kingdom) were used at a 1:1000 dilution. Secondary antibodies: goat anti-rabbit IgG, HRP-linked (7074; Cell Signaling Technology, Inc., Danvers, MA) and rabbit anti-rat IgG, HRP- linked (ab6734; Abcam, Cambridge, United Kingdom) were used at a 1:2500 dilution. Proteins were detected with an enhanced chemiluminescent substrate (34094; Thermo Fisher Scientific, Inc., Waltham, MA) and visualized on a UVP imaging system. For both donors tested, BCL11B protein was markedly higher in NK cells transfected with the BCL11B mRNA (FIG.11B). Together, these data demonstrate the efficacy of an mRNA approach for transcription factor overexpression. Example 5 Subset-specific impacts of BCL11B overexpression in NK cells CD56^bright NK cells proliferate at a significantly higher rate compared to CD56^dim NK cells in response to cytokine stimulation, and the developmental relationship between these subsets is unclear. Therefore, to determine the effects of Bcl11b overexpression on maturation and proliferation, NK cell subsets were sorted prior to transfection and ex vivo culture. First, CD56^bright peripheral blood NK cells were sorted and labeled with CELLTRACE (Life Technologies, Corp., San Diego, CA) dye to track proliferation. De-identified Trima cones were obtained from the Memorial Blood Center (Saint Paul, MN). Mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque Premium (Cytiva, Marlborough, MA). NK cells were then enriched from peripheral blood mononuclear cells using the EASYSEP Human NK cell enrichment kit (19055; STEMCELL Technologies, Inc., Vancouver, Canada) according to the manufacturer’s protocol. In some experiments, NK cells were labeled with CELLTRACE (Life Technologies, Corp., San Diego, CA) violet dye immediately after enrichment. NK cells were stimulated overnight in B0 media [Dulbecco’s modified Eagle’s media plus Ham’s F-12 media, 2:1, supplemented with 10% heat-inactivated human AB serum, penicillin (100 U/ml), streptomycin (100 µg/mL), ^-mercaptoethanol (25 mM), ascorbic acid (20 mg/ml), and sodium selenite (5 ng/ml)] with IL-15 (10 ng/ml; National Cancer Institute) at a density of 1.5×10⁶ cells/well in a 96-well U-bottom plate. These cells were transfected with mAzami control and BCL11B mRNAs and cultured for seven days with low-dose IL-15. Following overnight stimulation, NK cells were harvested and electroporated using a transfection system (4D-NUCLEOFECTOR, Lonza, Basel Switzerland) in a 16-well NUCLEOCURVETTE (Lonza, Basel Switzerland) strip in P3 buffer with program CM-137. Cells were either electroporated with no mRNA, 2 µg control mAzami mRNA, or 8 µg BCL11B mRNA. Transfected NK cells were then cultured in B0 media with IL-15 (1 ng/mL) at a density of 1×10⁶ cells/well in a 24-well plate for the remainder of the assays. Cells were harvested at days 1, 5, and 7 for phenotypic analyses of subset frequencies and proliferation by flow cytometry. Flow cytometry and cell sorting were performed as follows. For surface staining, NK cells were washed with flow buffer (1X PBS, 2% human AB serum, 2 mM EDTA) and incubated with fluorochrome-conjugated antibodies and LIVE/DEAD fixable near- IR dead cell stain kit (L34976; Invitrogen, Waltham, MA) for 30 minutes at 4 ^C. For intracellular staining after surface staining, cells were washed with flow buffer and then fixed with 2% paraformaldehyde. Cells were then permeabilized with permeabilization buffer eBioscience, San Diego, CA) and incubated with antibodies targeting intracellular proteins for 30 minutes at 4 ^C. Flow cytometry was performed on a flow cytometer (FACSYMPHONY A3, BD Biosciences, Franklin Lakes, NJ) and data was analyzed using FlowJo software (BD Biosciences, Franklin Lakes, NJ). Cell sorting was performed using a high sensitivity flow cytometer (FACSARIA, BD Biosciences, Franklin Lakes, NJ). Peripheral blood mononuclear cells were isolated as described above. CD3⁺ and CD19⁺ cells were depleted using EASYSEP the human CD19 positive selection kit II (17854; STEMCELL Technologies, Inc., Vancouver Canada) and the EASYSEP human CD3 positive selection kit II (17851; STEMCELL Technologies, Inc., Vancouver Canada). Cell staining was performed as described above. Cells were stained with combinations of the following anti-human antibodies: CD158 (339506; BioLegend, San Diego, CA), CD158b (312606; BioLegend, San Diego, CA), CD158e (312708; BioLegend, San Diego, CA), NKG2A (A60797; Beckman Coulter, Inc., Brea, CA), CD16 (302046; BioLegend, San Diego, CA), CD57 (393304; BioLegend, San Diego, CA), CDKN1A (8587S; Cell Signaling Technology, Inc., Danvers, MA), CDKN2C (SC-9965 PE; Santa Cruz Biotechnology, Inc., Dallas, TX), CDK2 (14174S; Cell Signaling Technology, Inc., Danvers, MA), CDK4 (42749S; Cell Signaling Technology, Inc., Danvers, MA), Granzyme B (372222; BioLegend, San Diego, CA), Perforin (308106; BioLegend, San Diego, CA), and Stat5 (pY694) (612599; BD Biosciences, Franklin Lakes, NJ). As CD56^bright NK cells transfected with BCL11B mRNA began to proliferate between days 1 and 5, the frequencies of mAzami positive cells dropped significantly. As proliferation rates increased between days 5 and 7, the frequencies of mAzami positive cells continued to decrease in cells that were transfected with BCL11B mRNA (FIG.12A). CELLTRACE (Life Technologies, Corp., San Diego, CA) assessment dye dilution showed significantly less cell division by CD56^bright NK cells transfected with BCL11B mRNA relative to non-transfected cells and cells transfected with the mAzami control mRNA (FIG.12B). NK cell differentiation is associated with a loss of NKG2A expression and sequential acquisition of CD16, killer immunoglobulin-like receptor (KIR), and CD57. Surface levels of these receptors were also analyzed by flow cytometry. CD56^bright NK cells transfected with BCL11B maintained expression of NKG2A and did not acquire CD16 or CD57. Moderate KIR expression was observed at day 7 but lower in cells expressing BCL11B (FIG.12A; FIG.27). Thus, BCL11B expression in CD56^brights severely limits proliferative responses to IL-15 without any discernable impact on maturation. Next, CD56^dimCD57- NK cells were sorted from peripheral blood and subjected to the same experimental setup described above. Similar to what was observed with sorted CD56^bright NK cells, mAzami frequencies declined dramatically over time in CD56^dimCD57- NK cells transfected with BCL11B mRNA (FIG.13), and this was associated with much lower rates of cell division for the CD16-CD57- and CD16⁺CD57- subsets (FIG.13). CD57 acquisition defines a late stage of human NK cell maturation and is inducible with IL-15 stimulation (Björkström et al., 2010. Blood.116:3853-3564). CD57 upregulation was observed after 5 days of culture on the surface of sorted CD56^dimCD57- NK cells transfected with both the mAzami control and BCL11B mRNAs. Importantly, the frequencies of CD57⁺ cells were higher in NK cells expressing BCL11B mRNA compared to those expressing control mRNA. An overall decrease in the percentages of CD57⁺ NK cells was observed at day 7 concomitant with increased proliferation of CD57- subsets, but relative frequencies remained higher in cells expressing BCL11B (FIG.14). No significant differences in subset distribution based on surface levels of NKG2A or KIR were observed between cells expressing control or BCL11B mRNAs (FIG.30). These data suggest that BCL11B slows down the proliferation of CD56^dimCD57- NK cells while simultaneously enhancing late-stage maturation driven by IL-15. The sharp drops in the frequencies of CD56^bright and CD56^dimCD57- NK cells expressing BCL11B mRNA throughout the seven-day culture period may have been due to either a slowing of proliferation or BCL11B mRNA being unstable over time after NK cell transfection. To address this issue, CD56^dimCD57⁺ NK were sorted cells from peripheral blood and the experiments were repeated. The CD56^dimCD57⁺ NK cell subset shows limited proliferation in response to IL-15, with only a small fraction achieving 1 cell division. Therefore, by transfecting these cells and culturing with IL-15, BCL11B mRNA stability could be assessed largely independent of proliferation. Modest decreases in mAzami frequencies were observed over time in CD56^dimCD57⁺ NK cells transfected with BCL11B mRNA that did not reach statistical significance (FIG.15). Overall proliferation rates were low and there was a trend towards more cells with BCL11B mRNA transfection exhibiting 0 divisions (FIG.15). No significant subset differences were observed between cells expressing control or BCL11B mRNA (FIG.31). Our data aligns with previous results showing that CD57⁺CD56^dim NK cells proliferate significantly less in response to cytokine stimulation relative to CD57-CD56^dim NK cells. To determine whether CD56^dimCD57⁺ cells are responsive to IL-15, NK cells were isolated from peripheral blood and STAT5 phosphorylation in subsets were measured by flow cytometry. pSTAT5 levels were highest in CD56^bright and CD56^dimCD16-CD57- NK cells. However, STAT5 phosphorylation was observed in CD56^dimCD16⁺ NK cells, and levels of phosphorylation were similar between CD56^dimCD16⁺CD57- and CD56^dimCD16⁺CD57⁺ NK cells (FIG.32). Together, these results suggest that the BCL11B mRNA is stable, and the relative decreases in CD56^bright and CD56^dimCD57- NK cells expressing BCL11B mRNA are due to inhibition of proliferation. Example 6 NK cells overexpressing BCL11B exhibit elevated levels of cyclin-dependent kinase inhibitors BCL11B may have a suppressive influence on cell cycle progression. Overexpression of BCL11B in T cell lines result in cell cycle restriction and correlated with upregulation of cyclin- dependent kinase inhibitors. Having observed reduced cell cycle progression due to BCL11B mRNA transfection in sorted subsets of primary NK cells, enriched CD3-CD56⁺ NK cells from peripheral blood were transfected with mAzami control or BCL11B mRNA and cultured for seven days with low-dose IL-15. Intracellular staining was performed with antibodies against cyclin-dependent kinase inhibitor 1A (CDKN1A), cyclin-dependent kinase inhibitor 2C (CDKN2C), cyclin-dependent kinase 2 (CDK2), or cyclin-dependent kinase 4 (CDK4). CDKN1A binds and inhibits the activity of CDK2 to negatively regulate the G1 to S phase transition of the cell cycle. CDKN2C binds CDK4 to limit cell cycle progression. In total CD3- CD56⁺ NK cells transfected with BCL11B mRNA, CDKN1A expression was significantly higher at the end of the culture period compared to cells transfected with control mRNA (FIG.16). When looking more specifically at NK cell subsets based on CD16 and CD57 expression, CDKN1A levels decreased across the spectrum of maturation but were significantly higher in all three subsets with BCL11B mRNA transfection (FIG.16). CDK2 levels were similar in total CD3-CD56⁺ NK cells transfected with control or BCL11B mRNAs (FIG.16) and in each gated subset (FIG.16). CDKN2C levels were also higher in total CD3-CD56⁺ NK cells overexpressing BCL11B (FIG.17) and in each subset (FIG.17). CDK4 levels mirrored those of CDK2, with little difference observed between control and BCL11B mRNA transfected bulk NK cells (FIG. 17) or NK cell subsets (FIG.17). Together, these data suggest that BCL11B limits NK cell proliferation through induction of cyclin-dependent kinase inhibitors. Example 7 Inhibition of cell division by Bcl11b is associated with increased levels of cytotoxic granule components Conditional knockout of Bcl11b in mature mouse CD8⁺ T cells is associated with reduced cytotoxicity and lower production of granzyme B and perforin. When CD3-CD56⁺ NK cells were transfected with BCL11B mRNA and cultured for seven days, a pronounced enrichment of cells with high forward scatter (FSC) was observed compared to NK cells transfected with mAzami control mRNA (FIG.19). FSC^high NK cells from all transfection conditions displayed elevated perforin and granzyme B compared to FSC^low cells, and significantly higher levels of both cytotoxic proteins were observed in FSC^high NK cells overexpressing BCL11B relative to controls (FIG.18; FIG.19). To examine this phenomenon in more depth, cell proliferation within the FSC^high and FSC^low subsets was investigated. Significant proportions of FSC^high NK cells overexpressing BCL11B did not divide after seven days of culture with IL-15 (FIG.19). Perforin and granzyme B levels were highest in FSC^high NK cells overexpressing BCL11B and decreased during each cell division (FIG.20). No differences in perforin or granzyme B levels were observed when comparing FSC^low subsets (FIG.21). Increased perforin and granzyme B levels were not simply a function of enhanced maturation, as both CD16⁺CD57⁺ and CD16⁺CD57- NK cell subsets exhibited significantly higher levels of these cytotoxic molecules after transfection with BCL11B relative to mAzami controls (FIG.32). Thus, BCL11B upregulates perforin and granzyme B in a manner that is inversely correlated with proliferation. Example 8 Induction of BCL11B in iPSC-derived NK cells enhances killing capacity and in vivo tumor control The reduced rates of proliferation by peripheral blood NK cells transfected with BCL11B in response to low-dose IL-15 made it difficult to determine the impact of BCL11B on NK cell function. To answer this question, a doxycycline-inducible BCL11B construct was designed that was inserted into the AAVS1 safe harbor locus of bone marrow-derived iPSCs using CRISPR/Cas9. Bone marrow iPSCs (IISH2i-BM9) were obtained from WiCell and maintained on MATRIGEL (Discovery Labware, Inc., Bedford, MA)-coated plates (Corning, Inc., Corning, NY) in mTeSR plus media (100-0276; STEMCELL Technologies, Inc., Vancouver, Canada). Human BCL11B open reading frame (NM_138576.4) was cloned into an AAVS1-transposon vector downstream of the TREtight promoter of the pTRE-P2A-Venus-CAG-puro plasmid and integrated into AAVS1 alleles using CRISPR-Cas9 as previously described (Oceguera-Yanez et al., 2016. Methods.101:43-55). Briefly, 1-2.5×10⁶ singularized human iPSCs were nucleofected with 6 µg AAVS1 donor plasmid along with AAVS1 guide RNA and Cas9 (Synthego Corp., Redwood City, CA) in 20 µL of P3 primary cell solution (4D-NUCLEOFECTOR; Lonza, Basel, Switzerland; V4XP-3032) using program CA-137. Nuclofected iPSCs were then plated into one well of a MATRIGEL (Discovery Labware, Inc., Bedford, MA)-coated six-well plate in 3 mL of prewarmed mTeSR media containing 10 µM Y27632 (1254; Tocris Bioscience, Bristol, United Kingdom). Twenty-four hours later, the media was replaced with fresh mTeSR media. When cells reached 80% confluency, 1 µg/ml puromycin was added for drug selection. Resistant clones were screened by Venus expression under a fluorescence microscope with doxycycline treatment. Selected iBCL11B-iPSC clones were expanded, and BCL11B integration into the AAVS1 locus was confirmed by genomic PCR. iPSCs containing the BCL11B construct were differentiated into CD34⁺ hematopoietic progenitor cells and then the cultures were split for NK cell differentiation. iBCL11B-iPSCs were differentiated towards the mesoderm and CD34⁺ hematopoietic progenitor stages in chemically defined serum-free media supplemented with mesoderm- inducible cytokines and hematopoietic cytokine cocktails (PeproTech, Inc., Chelmsford, MA). iCD34⁺ cells were subsequently enriched prior to differentiation. At the beginning of the iNK cell differentiation culture, iCD34⁺ cells were plated on OP9-DLL4 stromal cells in B0 media to support NK cell differentiation. To induce BCL11B during NK cell differentiation, doxycycline (2 µg/ml) was added to the culture media. After 20-30 days of culture, iNK cells were harvested and co-cultured with irradiated K562 cells transduced with membrane-bound IL-21 and 4-1BB ligand (4-1BBL) constructs in supplemented B0 media for one week. Half of the cells received doxycycline weekly during NK cell lineage specification, while the other half were untreated. At the end of differentiation, both sets of NK cells were expanded with engineered feeders to obtain enough cells for in vitro and in vivo testing. Interestingly, iNK cells treated with doxycycline were maintained at higher levels during differentiation and expanded at higher rates when co-cultured with feeders relative to iNK cells cultured without doxycycline (FIG.22). Consistent with observations using primary peripheral blood NK cells, iNK cells treated with doxycycline contained elevated levels of granzyme B and perforin (FIG. 22). The expression of granzyme B and perforin in iNK cells derived from Bcl11b-KO iPSCs was also examined. iNK cells from Bcl11b-KO iPSCs showed no expression of either granzyme B or perforin (FIG.32B). Cytotoxic effector functions were then tested in two-dimensional, real-time imaging experiments where iNK cells cultured with and without doxycycline were co-cultured with monolayers of A549 lung carcinoma (FIG.23) and SKOV3 ovarian adenocarcinoma (FIG.23) cells. An INCUCYTE S3 live cell imager (Sartorius AG, Göttingen, Germany) was used for real- time imaging of iNK cell and target cell cocultures. The lung carcinoma cell line A549 (CLL- 185; ATCC) and ovarian adenocarcinoma cell line SKOV3 (HTB-77; ATCC) were cultured in DMEM media or RPMI media, respectively, supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. Both cell lines were transduced with Nuclight Red (Sartorius AG, Göttingen, Germany) for visualization. For 2-D assays, target cells were plated in 96-well flat- bottom optical plates (Thermo Fisher Scientific, Inc., Waltham, MA) at a density of 1×10³ cells per well. Cells were cultured overnight to allow a monolayer to form. The next day, iNK cells were added in triplicate at 4:1 E:T ratios in B0 media supplemented with 10 ng/ml IL-15. Plates were subsequently transferred to the live cell imager and scanned hourly for 72 hours. For 3-D assays, 2×10³ target cells were added to each well of 96-well ultra-low adherence plates (Corning, Inc., Corning, NY) and cultured for 48 hours to allow for spheroid formation. Following spheroid stabilization, iNK cells were added in triplicate at 4:1 E:T ratios in the same media conditions described above. Complete plates were placed in the live cell imager culture chamber and scanned every hour for 120 hours. For 2-D assays, the “Red Counts Per Image” metric was used to quantify live target cell present. For 3-D assays, the “Largest Red Object Area” metric was used to quantify spheroid size. To normalize sample metrics, raw sample data was adjusted to the mean of the target alone control metrics at each time point and then subsequently adjusted to corresponding normalized sample metric values at time point 0. iNK cells treated with doxycycline mediated significantly faster and more robust cytotoxicity against both cell lines relative to control iNK cells that did not receive doxycycline. Similar experiments were then performed where A549 and SKOV3 tumor cells were plated and allowed to form spheroids in culture prior to the addition of iNK cells. In these three-dimensional co-culture assays, more rapid and complete killing was observed in iNK cells treated with doxycycline (FIG.24). Having tested the cytotoxicity of iNK cells overexpressing BCL11B in vitro, their in vivo efficacy against solid tumors was investigated. Using NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/Szj (NSG) mice (005557; The Jackson Laboratory), a xenogenic model of ovarian cancer was used. Mice were balanced into groups based on initial bioluminescence imaging (BLI), and groups of mice received no treatment (tumor alone) or intraperitoneal (i.p) injections of immediately thawed control iBCL11B iNK cells (no doxycycline) or iBCL11B iNK cells (doxycycline-treated) at a dose of 1×10⁷ per mouse. IL-2 was i.p injected three times per week for three weeks to support iNK cell survival, and tumor BLIs were measured weekly for the duration of IL-2 support.8-10- week-old female NSG mice (n = 12) were injected i.p. with 1×10⁵ luciferase-expressing OVCAR8 ovarian cancer cells, and tumors were established over three days. BLIs were then performed, and mice were balanced into three groups (n = 4 mice per group) based on the initial BLI measurements. The first group of mice did not receive any treatment. The second group of mice received an i.p. injection of 1×10⁷ iBCL11B iNK cells that were not cultured with doxycycline during differentiation. The third group received an i.p. injection of 1×10⁷ iBCL11B iNK cells that were differentiated in the presence of doxycycline. All iNK cells were thawed from cryopreservation, washed, and reconstituted in PBS prior to injection. All mice receiving iNK cells were also given i.p. injections of IL-2 (5×10⁴ U/mouse) three times per week for three weeks to support iNK cell survival. Bioluminescence imaging was performed weekly for three weeks, after which the mice were monitored for survival. At day 21, the average tumor burden was six-fold lower in mice treated with control iBCL11B iNK cells (no doxycycline) and 21-fold lower in mice treated with iBCL11B iNK (doxycycline-treated) cells relative to the tumor alone group. Average tumor burden was three- fold lower in the doxycycline-treated iBCL11B iNK cell treatment group relative to the control iNK cell treatment group (FIG.25). Mice treated with iBCL11B iNK cells also survived significantly longer (mean 68 days) compared to mice with tumor alone (mean 49 days) and mice treated with control iNK cells (mean 52.5 days) (FIG.26). Collectively, these data show that BCL11B overexpression in iNK cells supports superior cytotoxic function and tumor control in vivo. Statistical analyses described were performed using PRISM software (GraphPad Software Inc., San Diego, CA). Student’s t-tests were used when comparing two groups with normal-distributed data. Two-way ANOVA tests were used to determine statistical significance in datasets comparing two groups with a time-dependent variable. When comparing more than two groups, one-way ANOVA with Dunnet’s multiple comparison analysis for normal- distributed data was used. Example 9 Interrogation of T cells iBCL11B-iPSCs were differentiated towards the mesoderm and CD34⁺ hematopoietic progenitor stages in chemically defined serum-free media supplemented with mesoderm- inducible cytokines and hematopoietic cytokine cocktails (PeproTech, Inc., Chelmsford, MA). iCD34⁺ cells were subsequently enriched prior to differentiation. At the beginning of the iT cell differentiation culture, iCD34⁺ cells were plated on OP9-DLL4 stromal cells in OP9 medium (MEMα with 20% FBS), supplemented with TPO (10-20 ng/ml), SCF (20-30 ng/ml), 10 ng/ml IL-7 (5-15 ng/ml) and Flt3L (5-15 ng/ml) for 3-4 weeks to complete T lineage commitment. Differentiating T lymphoid cells were passaged onto fresh OP9-DLL4 monolayers every seven days and fresh media supplemented with cytokines were added 48 hours after each passage. To induce BCL11B during iT cell differentiation, doxycycline (2 µg/ml) was added to the culture media. After 3-4 weeks of culture, iT cells were harvested checked for the expression of TCR ^ ^ and bar graph was plotted (FIG.32A) for expression of TCRαβ ^from three independent experiments. The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified. Sequence Listing Free Text SEQ ID NO:1- positive genotyping primer 1 CTGTTTCCCC TTCCCAGGCA GGTCC SEQ ID NO:2- positive genotyping primer 2 TCGTCGCGGG TGGCGAGGCG CACCG SEQ ID NO:3- homozygous genotyping primer 1 CGGTTAATGT GGCTCTGGTT SEQ ID NO:4- homozygous genotyping primer 2 GAGAGAGATG GCTCCAGGAA SEQ ID NO:5- GAPDH qPCR fwd GGAGCGAGAT CCCTCCAAAA T SEQ ID NO:6- GAPDH qPCR rev GGCTGTTGTC ATACTTCTCA TGG SEQ ID NO:7- BCL11B qPCR fwd CTTCAACAGC GCATGGTTCC SEQ ID NO:8- BCL11B qPCR rev GAAGTTCATC AGCGGGCTCT

Claims

What is claimed is: 1. A method of modulating hematopoietic differentiation in a stem cell, the method comprising inducing expression of BCL11B in the stem cell and differentiating the stem cell to form a differentiated cell.

2. The method of claim 1, wherein the differentiated cell comprises a lymphoid cell.

3. The method of claim 2, wherein the lymphoid cell is an NK cell.

4. The method of claim 3, wherein the NK cell is a CD56^bright NK cell.

5. The method of claim 2, wherein the lymphoid cell is a T cell.

6. The method of claim 5, wherein the T cell is a CD8⁺ T cell.

7. The method of any preceding claim, wherein expression of BCL11B is induced during the mesoderm stage of hematopoietic differentiation.

8. The method of any preceding claim, wherein expression of BCL11B is induced during NK cell differentiation.

9. The method of any preceding claim, wherein inducing expression of BCL11B comprises inducing expression and subsequently stopping induction.

10. The method of any preceding claim, wherein the method increases formation of arterial hemogenic endothelial (AHE) cells compared to a stem cell in which expression of BCL11B is not induced.

11. The method of any preceding claim, wherein the method increases formation of multipotent hematopoietic progenitor (MHP) cells compared to a stem cell in which expression of BCL11B is not induced.

12. The method of any preceding claim, wherein the method increases NK cell cytotoxicity compared to an NK cell differentiated from stem cells in which expression of BCL11B is not induced.

13. The method of any preceding claim, wherein the stem cell is an induced pluripotent stem cell (iPSC) or a hematopoietic stem cell (HSC).

14. The method of any preceding claim, wherein BCL11B expression is induced in the stem cell in vitro.

15. The method of any preceding claim, further comprising isolating the differentiated cell.

16. A nucleic acid including a sequence of BCL11B and an inducible promoter, wherein expression of BCL11B is controlled by the inducible promoter.

17. The nucleic acid of claim 16, further comprising a selectable marker.

18. A gene expression vector comprising the nucleic acid of claim 16 or claim 17.

19. An mRNA produced by the nucleic acid of claim 16, wherein the mRNA comprises the open reading frame of BCL11B.

20. An engineered cell comprising the nucleic acid of any preceding claim.

21. The engineered cell of claim 20, wherein the cell is a stem cell, such as an iPSC or an HSC.

22. The engineered cell of claim 20 or claim 21, wherein the nucleic acid is integrated into the genome of the cell.