Pluripotent cell differentiation

Abstract

This invention provides improved methods for differentiating human pluripotent stem cells into an aorta-gonad-mesonephros-like definitive hemogenic mesoderm capable of giving rise to definitive hematopoietic progenitor and stem cells.

Description

PLURIPOTENT CELL DIFFERENTIATIONREFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of US Provisional Application No. 63 / 545,916, filed October 26, 2023, which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] Incorporated by reference in its entirety is a computer-readable amino acid sequence listing submitted concurrently herewith and identified as follows: 17,756 bytes XML file named 10466-W001-SEC_Sequence_Listing; created on October 22, 2024.FIELD OF THE INVENTION

[0003] The present invention relates to pluripotent cell differentiation and constructs utilizing this.BACKGROUND OF THE INVENTION

[0004] Hematopoietic cells or blood cells are in great demand for clinical applications and for laboratory use. In the clinic, hematopoietic stem cells (HSCs) can be used to reconstitute hematopoiesis in patients that have undergone a therapy that suppresses hematopoiesis, such as an anti-cancer therapy, or in patients that have inherited hematological diseases. In addition, red blood cells, platelets, and neutrophil granulocytes can be used in blood transfusions and in the treatment of certain hematological disorders. In the lab, blood cells can be used for many applications including drug screening.

[0005] Human pluripotent stem cells are a tremendous tool to model early human development and disease including their use in the in vitro generation of blood cell fates. Hematopoietic progenitors and stem cells are the primary source of blood and the immune system from early development to adulthood and arise through successive waves of hemogenic mesoderm either in the yolk sac or embryo proper. Researchers have long sought a tractable human model for observing and distinguishing these waves of hematopoiesis in the dish for human developmental and disease modeling.

[0006] Hematopoietic stem cells (HSCs) give rise to all cell lineages of the blood. This provides a continuous lifetime supply of mature blood cells and can reconstitute the whole hematopoietic system of a conditioned recipient after infusion. Recent progressdemonstrates that human induced pluripotent stem cells (HiPSC) are able to be differentiated into HSC which has long-term engraftment capability. The development of hematopoiesis is comprised of multiple, partially overlapping but spatiotemporally separated waves. Definitive hematopoiesis generating HSC that give rise to all mature blood and immune cells is generated in a third wave from hemogenic endothelium (HE) cells emerging from the aorta gonad-mesonephros (AGM) region through a process called endothelial-to-hematopoietic transition (EHT).

[0007] We have developed a protocol to produce functional human HSC from HiPSC. This novel protocol mimics the definitive hematopoietic development wave, first induce the iPSC into AGM-like hemogenic endothelium (PCT Application Number WO202 1097346). The HSCs can then be further efficiently differentiated into macrophages, dendritic cells, and erythrocytes.

[0008] Macrophages are pivotal effectors of host immunity and regulators of tissue homeostasis. Human macrophage biology has been hampered by the lack of reliable and scalable models with current state-of-art being peripheral blood mononuclear cells (PBMCs). Human induced pluripotent stem cell (hiPSC)-derived monocytes and macrophages, offer a better alternative to PBMCs, serving as an unlimited source of subject genotype-specific cells.

[0009] Macrophages have become increasingly relevant in the immune-oncology space, particularly in the treatment of solid tumors. Macrophages can be stimulated to induce a Ml state, making the tumor microenvironment pro-inflammatory, allowing for efficient recruitment of effector immune cells to enter the tumors. Additionally, macrophages have been shown to possess the ability to phagocytose cancer cells, given the right conditions. iPSC-derived macrophages present an opportunity to provide an ample source of these cells for therapeutic purposes.

[0010] Currently, blood cells for such clinical and laboratory applications are obtained from living donors. However, the limited supply of donor blood, especially when a genetically- compatible donor is required, limits therapeutic applications and drug screening. Thus, there remains a need to develop sources of blood cells other than donor blood. For example, there is a need for an unlimited supply of well characterized functional blood cell types, including patient specific HSCs for therapeutic applications.SUMMARY OF THE INVENTION

[0011] The present invention is based, in part, on the discovery of a method of producing macrophages from hiPSC.BRIEF DESCRIPTION OF THE FIGURES

[0012] Figure 1 shows FACS plots showing hemogenic endothelium formation from iPSC, using an earlier protocol as well as the protocol shown in Example 1.

[0013] Figure 2 shows generation of HSC-like cells from iPSC-derived hemogenic endothelium cells at day 21.

[0014] Figure 3 shows the results of a limiting dilution assay at day 21 of differentiation. Figure 3A shows the percent of the wells having each cell type when the wells were loaded with a different number of cells. Figure 3B shows the number of colonies formed of different cell types following loading by a different number of cells.

[0015] Figure 4 shows the generation of GFP-2A-Runxlc hiPSC reporter line for labeling of hematopoietic stem cells (HSCs). Figure 4A shows a schematic picture showing the strategy to target Runxlc genomic locus: Runxlc is transcribed from the distal promoter with a unique exon. Guide RNA was designed to specifically target the ATG start codon of Runxlc transcript for precise genome editing. A GFP-2A sequence was fused at the N- terminus to fluorescently label Runxlc positive hematopoietic stem cells during differentiation. The LoxP-PGK-BSD-pA-LoxP selection cassette was place in intron 1 to facilitate enrichment of correctly targeted cells populations. PCR primers (see Table 1) were designed to amplify the left junction of homologous recombination and the GFP-2A- Runxlc linker sequence. Figure 4B shows that the primers described in 4A was used for screening positive colonies after genome editing. After blasticidin selection, a total of 48 single cell clones were picked, expanded and subjected PCR genotyping analysis. 38 clones exhibited positive genotyping band on agarose gel (efficiency = 79%). Figure 4C shows an image of the selected positive clone of GFP-2A-Runxlc hiPSC line.

[0016] Figure 5 shows a visualization of GFP positive hematopoietic stem cells in hiPSC differentiation: GFP-2A-Runxlc iPSCs (dO, top left panel) were firstly differentiated into endothelium (d9, top right panel), followed by induction of endothelial- hematopoietic transition (EHT) that results in emergence of GFP positive hematopoietic stem cells (dl4, mid panel) from selected regions (dashed box, “blood island”) of GFP negative endothelial layer. At day 17, the production of GFP positive HSCs are no longerrestricted in certain regions, but became more prominent throughout the tissue culture (dl7 bottom panel).

[0017] Figure 6 shows a time course of surface marker expression pattern of GFP- 2A-Runxlc iPSCs during hematopoietic differentiation: (A) Single positive population. (B) Runxlc+CD34+CD45+ putative hematopoietic stem cell population.

[0018] Figure 7 shows HSC CD34 vs GFP-Runxlc expression on days 9 and 14.

[0019] Figure 8 shows HSC CD34 vs GFP-Runxlc expression on days 16 and 17.

[0020] Figure 9 shows HSC CD34 vs GFP-Runxlc expression on days 20 and 21.

[0021] Figure 10 shows cell population sorting for CFU assays from LT-iPSC andGFP-Runxlc iPSC.

[0022] Figure 11 shows CFU total cell counts on days 16, 17, 20, and 21.

[0023] Figure 12 shows a CFU panel of common progenitor markers on days 16,17, 20, and 21.

[0024] Figure 13 shows a CFU panel of lymphoid progenitor markers on days 16, 17, 20, and 21.

[0025] Figure 14 shows a CFU panel of myeloid progenitor markers on days 16, 17, 20, and 21.

[0026] Figure 15 shows: (A): Schematic of the differentiation of LT iPSC to definitive hematopoietic stem cells (HSCs). In short, iPSCs were plated as single cells and then inducted to undergo developmental-stage specific differentiation by first mesoderm induction, followed by endothelial formation. Lastly, a specified cocktail was added so that differentiation cultures undergo endothelial-to-hematopoietic transition and subsequent maturation; (B): Brightfield images of day 0 (DO) iPSCs, hemogenic endothelium on day 9 (D9), and floating iPSC derived HSCs on day 18-20 (DI 8-20); (C): Representative flow cytometry plots show the gating strategy for identifying HSCs from iPSC differentiation cultures. Here, we characterize HSCs as lin-CD45+CD34+CD45RA-CD38- / lowCD90+; (D): Brightfield images overlayed with GFP channel showing green fluorescence in budding cells undergoing EHT; (E): Representative flow cytometry plot showing the RUNXlc GFP expression in CD34+CD45+ cells from differentiation cultures; (F): Schematic describing the methylcellulose and limiting dilution assays to assess the potency of the iPSC derived HSCs; (G): Representative brightfield images of various colonies found in CFU assays. Colonies were counted and categorized as BFU-E, CFU-E, CFU-G, CFU- M, CFU-GM, and CFU-GEMM. Quantifications are shown as both pie charts and bar graphs; (H): Quantification of CFUs at various input cell numbers for limiting dilutionanalysis. CFC frequency was calculated based by calculating the number of cells necessary to achieve colonies in 37% of wells or ~1 in 5 cells.

[0027] Figure 16 shows: (A): qRT-PCR analysis of mesoderm-, endothelial-, artery- , reprogramming-, and AGM-specific genes through the course of iPSC differentiation towards a definitive HSC; (B): Representative flow cytometry plot of day 3 (D3) iPSC differentiation cultures measuring the expression of cell surface markers CXCR4 and KDR / FLK1; (C): Day 3 expression of CXCR4 and KDR / FLK1 from the differentiation of the TBXT EGFP reporter line; (D): Expression of TBXT EGFP (red) at day 3 of differentiation compared to the parental LT iPSC line (blue); (E): Representative flow cytometry plots at day 3 of differentiation utilizing both the LT and TBXT EGFP line showing expression on cell surface markers CXCR4 and KDR / FLK1. Shading of the plots displays EGFP expression intensity within the four specified populations; (F): Quantification of the median fluorescent intensity within CXCR4-KDR / FLK1-, CXCR4+KDR / FLK1-, CXCR4+KDR / FLK1+, and CXCR4-KDR / FLK1- populations.

[0028] Figure 17 shows: (A): UMAP projection of the diffusion map (dmap) dimensionality reduction embeddings of the top 45 dimensions of the various induced pluripotent stem cell (iPSC) derived endothelial, hemogenic endothelium aorta-gonad- mesonephros (AGM)-like, pre-hematopoietic stem cell (pre-HSC), HSC, multipotent progenitor (MPP)-like, and blood cells. Additionally, human fetal liver (FL), umbilical cord blood (UCB), and bone marrow (BM) derived bona fide HSC’s were also included in he analysis. These data represent approximately 25% of that data (n=32,754 cells). F-statistic variance modeling was used to filter out noisy or lowly expressed genes, yielding 11,804 features that are stably expressed. Cells were sorted based on morphology, cell attachment, and hematopoietic markers (CD34, CD45, and CD90); (B): Single population highlights of UMAP projection for the various iPSC derived populations throughout the course of differentiation; (C): Bona fide human HSCs from three independent tissue sources. Each population contains biological and technical triplicates; (D): Dot plot displaying the expression of key stage-specific hematopoietic and developmental genes across specified iPSC derived clusters and bona fide HSCs. Color represents the average expression, and the size of the dot indicates the percent of cells within the cluster expressing specified genes; (E): Dot plot of cell-type identification through gene expression utilizing Seurat’s module score feature. Expression of gene modules is scored based on the average expression and percent of the population that expresses the set of given genes; (F): Feature plots displaying the expression of key hemogenic genes established by the field that isexpressed during distinct stages of development; (G): UMAP projection of the RNA velocity was calculated using spliced and unspliced counts to solve transcriptional dynamics.

[0029] Figure 18 shows: (A): Schematic of differentiation approach to testing the differences between our iPSC derived definitive HSCs with that of published primitive protocols with and without polarizing agents; (B): Macrophages were differentiated from human monocytes (CD14+), iPSC derived HSCs, and HSCs from CD34+ umbilical cord blood. The table summarizes the expression of MO (naive) and polarized macrophage (Ml and M2) markers assessed by flow cytometry; (C): Flow cytometry plots comparing the expression of classical and polarized macrophages from PBMCs and iPSCs; (D): Table summarizing the yield, donor variability, and percentage of both pan- and classical- monocyte markers; (E): MSD cytokine analysis of MO, Ml, and M2 polarized macrophages from primitive and definitive iPSC derived HSCs.

[0030] Figure 19 shows: (A): Experimental approach for the generation of HER2 CAR macrophages by lentiviral transduction of the CAR in iPSCs; (B): HER2 antigen binding in transduced HER2 CAR iPSC and the GFP CAR control. Unstained and stained iPSCs that do not have the CAR insertion were used as negative controls for unspecific antigen binding; (C): Representative flow plots showing expression of CD34+ and CD45+ after untransduced and HER2 CAR transduced iPSC lines were differentiated to definitive HSC. Expression of the CAR was confirmed by HER2 antigen binding; (D): The efficacy of iPSC derived untransduced and HER2 CAR transduced macrophages to phagocytosis SKOV3 HER2+ cancer cells was assessed by labeling macrophages with DAPI (CMAC) and SKOV3 (CMTPX) with TRICTC Cell Tracker dyes. Cells were mixed at a 10:1 (Effector: target) ratio for 24 and 48 hours. Doubles-positive cells were assessed by flow cytometry; (E): Quantification of Figure D by calculation percent specific lysis; (F): Enriched gene ontology plot of the biological process of genes that are upregulated in iPSC derived HER2 CAR Ml macrophages cultured with SKOV3 versus HER2 CAR Ml macrophages. Approximately 98 genes were upregulated with a p-value < 0.05 and log2FC > 1.

[0031] Figure 20 shows: (A): Experimental approach for the generation of T-cells from iPSC derived definitive HSCs using artificial thymic organoids (ATO); (B): Brightfield images at various magnifications of the same field after 4 weeks of differentiation to T-cells using both cord blood and iPSC derived HSCs in ATOs; (C): Representation flow plots showing the expression of CD3 and TCRab double positivepopulations (top), along with CD4 and CD8 expression (bottom) from ATO differentiations gated on the total number of cells; (D): Same plots as Figure 6C, except gates were set to only include cells that were CD3+. Expression of CD4 and CD8 were assessed within the total CD3+ population; (E): Flow cytometry flow showing the expression of classical T cell markers, such as CD4, TCRab, CD7, and CD8 gated on total viable cells. Expression patterns of these cell surface markers were compared between cord blood to iPSC derived HSCs in the ability of these cells to give rise to T cells in ATO cultures; (F): Flow cytometry of peripheral blood from NSG-SGM3 immunodeficient mice grafted with either CD34+ cord blood (CB) or iPSC derived HSCs at 12 weeks post-injection. Multilineage engraftment was observed (huCD45), including the presence of T-cells (huCD3), B-cells (huCD20), monocytes (huCD14), and NK-cells (huCD56) based on their respective cell surface markers. Two out of five animals grafted with the iPSC derived HSCs are shown and had iPSC derived peripheral blood contribution.

[0032] Figure 21 shows: (A): Targeting strategy for generating RUNXlc reporter iPSC lines; (B): Targeting strategy for the generation of TBXT EGFP LT iPSC reporter line; (C): Control flow plots showing DAPI and TRITC fluorescence with the specified cell types without co-culture. Day 0 (DO) cultures were trypsinized and mixed immediately prior to acquisition.

[0033] Figure 22 shows: (A): Flow cytometry plots of peripheral blood from remaining NSG-SGM3 within the experimental cohort. Chimerism was assessed using a human specific CD45 antibody (clone H130). Animal cohorts include those grafted with 3x104 umbilical cord blood CD34+ cells (left), freshly cultured iPSC-derived HSCs (middle), and lastly iPSC-derived HSCs that were cryopreserved prior to engraftment (right).DETAILED DESCRIPTION OF THE INVENTION

[0034] We have developed a high-efficiency method for differentiating human pluripotent stem cells into an aorta-gonad -mesonephros-like definitive hemogenic mesoderm capable of giving rise to definitive hematopoietic progenitor and stem cells. The hematopoietic progenitor and stem cells exhibit robust multilineage in vitro colony forming potential. Gene expression analysis and single cell sequencing strongly support the developmental timing and notion that the pluripotent stem cell derived hematopoietic stem and progenitors appear to have properties of bone fide hematopoietic stem cells. Thehematopoietic progenitors can be subsequently differentiated into polarized macrophage and T-cells in vitro. Minimal silencing was observed upon differentiation of the pluripotent stem cells to hematopoietic lineages when conducting gene editing, and upon engraftment into immunodeficient animals the hematopoietic progenitors and stem cells differentiate into multiple lineages including B-cells, T-cells, NK-cells, and monocytes.

[0035] In addition, we have developed methods to enhance iPSC-derived macrophage anti-tumor activity by generating iPSC lines that are transduced, and stably express, a chimeric antigen receptor (CAR) against a breast cancer antigen.

[0036] We have developed protocols to readily manufacture macrophages from hiPSC through definitive hematopoiesis paths based on HSC generated from our internal protocol. We have optimized the protocol to manufacture large scaler macrophages from hiPSC, including TC dish surface test, xeno-free medium conditions, freeze point test. Macrophages manufactured are fully characterized and functionally tested. Macrophage produced are comparable to human PBMC differentiated macrophages in term on morphology, surface markers expression, polarization (evaluated by markers and cytokine release). We also highlight the application of hiPSC-derived monocytes and macrophages as a gene-editing platform for functional validation in research and drug screening.

[0037] Recently, pluripotent stem cell lines have been obtained from human fibroblasts through insertion of certain genes critical for the maintenance of pluripotency of hESCs (Yu, I, et al. 2007, Science. 318: 1917-1920. Takahashi, K., et al. 2007, Cell. 131 :861-872. Park, I. H., et al. 2008, Nature. 451 : 141-146.). These so-called human induced pluripotent stem cells (iPSCs) behave similarly to hESCs, i.e., they are capable of self-renewal and large-scale expansion and differentiation toward all three germ layers. The hope is that iPSC lines generated from patients with various diseases could be used to obtain any type of progenitor or differentiated cell carrying a particular genetic trait at the cellular level, thus providing a unique opportunity to analyze disease pathogenesis in vitro.

[0038] Previously, a system was established for hematopoietic differentiation of hESCs into hematopoietic cells through coculture with OP9 bone marrow stromal cells (Vodyanik, M. A., Bork, J. A., Thomson, J. A., Slukvin, 1.1. 2005, Blood. 105:617-626) and characterized the two subpopulations of the most primitive multipotent hematopoietic cells to appear in OP9 cocultures of hESCs on the basis of their common expression of CD43 and differential expression of CD45. The lin-CD34+CD43+CD45- cells with broad lymphomyeloid differentiation potential appear first in coculture. Later, lin- CD34+CD43+CD45+ cells enriched in myeloid progenitors emerge (Vodyanik, M. A.,Thomson, J. A., Slukvin, 1. 1. 2006, Blood. 108:2095-2105.). The Slukvin lab demonstrated that a similar pattern of hematopoietic differentiation is observed when iPSCs differentiate into blood cells in coculture with OP9 (Choi, K., et al. 2009, Stem Cells. 27:559-567.).

[0039] In certain embodiments of the invention, there are disclosed methods and compositions for providing hematopoietic cells or precursors of hematopoietic cells by forward programming of human pluripotent cells that are not hematopoietic cells, including stem cells, which includes human embryonic stem cells and inducible pluripotent stem cells, or by transdifferentiation of somatic cells that are not hematopoietic cells. Also provided are cells that comprise exogenous expression cassettes including one or more hematopoietic precursor programming factor genes and / or reporter expression cassettes specific for hematopoietic cell or hematopoietic precursor cell identification. In some embodiments, the cells may be stem cells, including but not limited to, embryonic stem cells, fetal stem cells, or adult stem cells. In further embodiments, the cells may be any somatic cells.

[0040] Stem cells are cells found in most, if not all, multi-cellular organisms. They are characterized by the ability to renew themselves through mitotic cell division and the ability to differentiate into a diverse range of specialized cell types. The two broad types of mammalian stem cells are: embryonic stem cells that are found in blastocysts, and adult stem cells that are found in adult tissues. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells, and also maintain the normal turnover of regenerative organs, such as blood, skin or intestinal tissues.

[0041] Human pluripotent stem cells (including Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)) are capable of long-term proliferation in vitro, while retaining the potential to differentiate into all cell types of the body, including hematopoietic cells and hematopoietic precursor cells. Thus, these cells could potentially provide an unlimited supply of patient-specific functional hematopoietic cells and hematopoietic precursor cells for both drug development and therapeutic uses. The differentiation of human ESCs / iPSCs to hematopoietic cells and hematopoietic precursor cells in vitro recapitulates normal in vivo development; i.e. they undergo the normal sequential developmental stages including mesoderm differentiation and hematopoietic specification. That sequential developmental process requires the addition of different growth factors at different stages of differentiation. Certain aspects of the invention providefully functional hematopoietic precursor cells by forward programming from human ESCs / iPSCs or transdifferentiation from somatic cells via expression of a combination of transcription factors important for hematopoietic cell differentiation / function, similar to the generation of iPSCs, bypassing most-if not all-normal developmental stages. This approach may be more time- and cost-efficient, and generate hematopoietic precursor cells and hematopoietic cells with functions highly similar, if not identical, to human adult hematopoietic cells and precursors of hematopoietic cells. In addition, human ESC / iPSCs, with their unlimited proliferation ability, may be advantageous over somatic cells as the starting cell population for hematopoietic precursor cell differentiation. Examples of hematopoietic cells and precursors of hematopoietic cells produced as part of the invention include cells expressing CXCR4, cells that are CD34+,CD45+, CD90+ and THY1+, cells that are CD38-, Lin-, CD43- or CD73-, cells that are CD45+, CD34+, CD90+, CD38-, and Lin-, cells expressing CD90, cells expressing runxlc, or any combination of the above.

[0042] Embryonic stem cell lines (ES cell lines) are cultures of cells derived from the epiblast tissue of the inner cell mass (ICM) of a blastocyst or earlier morula stage embryos. A blastocyst is an early stage embryo-approximately four to five days old in humans and consisting of 50-150 cells. ES cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta.

[0043] Most research to date used mouse embryonic stem cells (mES) or human embryonic stem cells (hES). Both have the essential stem cell characteristics, yet they require very different environments in order to maintain an undifferentiated state. Mouse ES cells may be grown on a layer of gelatin and require the presence of Leukemia Inhibitory Factor (LIF). Human ES cells could be grown on a feeder layer of mouse embryonic fibroblasts (MEFs) and often require the presence of basic Fibroblast Growth Factor (bFGF or FGF-2). Without optimal culture conditions or genetic manipulation (Chambers et al., 2003), embryonic stem cells will rapidly differentiate.

[0044] A human embryonic stem cell may also be defined by the presence of several transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and Sox-2 form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency (Boyer et al., 2005). Cell surfaceantigens commonly used to identify hES cells include the glycolipids SSEA3 and SSEA4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81.

[0045] Human ES cells can be obtained from blastocysts using previously described methods (Thomson et al., 1995; Thomson et al., 1998; Thomson and Marshall, 1998; Reubinoff et al, 2000.) In one method, day-5 human blastocysts are exposed to rabbit antihuman spleen cell antiserum, then exposed to a 1 : 5 dilution of Guinea pig complement to lyse trophectoderm cells. After removing the lysed trophectoderm cells from the intact inner cell mass, the inner cell mass is cultured on a feeder layer of gamma-inactivated mouse embryonic fibroblasts and in the presence of fetal bovine serum. After 9 to 15 days, clumps of cells derived from the inner cell mass can be chemically (i.e. exposed to trypsin) or mechanically dissociated and replated in fresh medium containing fetal bovine serum and a feeder layer of mouse embryonic fibroblasts. Upon further proliferation, colonies having undifferentiated morphology are selected by micropipette, mechanically dissociated into clumps, and replated (see U.S. Pat. No. 6,833,269). ES-like morphology is characterized as compact colonies with apparently high nucleus to cytoplasm ratio and prominent nucleoli. Resulting ES cells can be routinely passaged by brief trypsinization or by selection of individual colonies by micropipette. In some methods, human ES cells can be grown without serum by culturing the ES cells on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor (Amit et al., 2000). In other methods, human ES cells can be grown without a feeder cell layer by culturing the cells on a protein matrix such as matrigel or laminin in the presence of "conditioned" medium containing basic fibroblast growth factor (Xu et al., 2001). The medium is previously conditioned by coculturing with fibroblasts.

[0046] Another source of ES cells are established ES cell lines. Various mouse cell lines and human ES cell lines are known and conditions for their growth and propagation have been defined. For example, the mouse CGR8 cell line was established from the inner cell mass of mouse strain 129 embryos, and cultures of CGR8 cells can be grown in the presence of LIF without feeder layers. As a further example, human ES cell lines Hl, H7, H9, H13 and H14 were established by Thompson et al. In addition, subclones H9.1 and H9.2 of the H9 line have been developed. It is anticipated that virtually any ES or stem cell line known in the art may be used with the present invention, such as, e.g., those described in Yu and Thompson (2008) Genes Dev 22(15): 1987-97, which is incorporated herein by reference.

[0047] The source of ES cells for use in connection with the present invention can be a blastocyst, cells derived from culturing the inner cell mass of a blastocyst, or cells obtained from cultures of established cell lines. Thus, as used herein, the term "ES cells" can refer to inner cell mass cells of a blastocyst, ES cells obtained from cultures of inner mass cells, and ES cells obtained from cultures of ES cell lines.

[0048] Induced pluripotent stem (iPS) cells are cells that have the characteristics of ES cells but are obtained by the reprogramming of differentiated somatic cells. Induced pluripotent stem cells have been obtained by various methods. In one method, adult human dermal fibroblasts are transfected with transcription factors Oct4, Sox2, c-Myc and Klf4 using retroviral transduction (Takahashi et al., 2007). The transfected cells are plated on SNL feeder cells (a mouse cell fibroblast cell line that produces LIF) in medium supplemented with basic fibroblast growth factor (bFGF). After approximately 25 days, colonies resembling human ES cell colonies appear in culture. The ES cell-like colonies are picked and expanded on feeder cells in the presence of bFGF.

[0049] Based on cell characteristics, cells of the ES cell-like colonies are induced pluripotent stem cells. The induced pluripotent stem cells are morphologically similar to human ES cells, and express various human ES cell markers. Also, when grown under conditions that are known to result in differentiation of human ES cells, the induced pluripotent stem cells differentiate accordingly. For example, the induced pluripotent stem cells can differentiate into cells having hematopoietic cell structures and hematopoietic cell markers. It is anticipated that virtually any iPS cells or cell lines may be used with the present invention, including, e.g., those described in Yu and Thompson, 2008.

[0050] In another method, human fetal or newborn fibroblasts are transfected with four genes, Oct4, Sox2, Nanog and Lin28 using lentivirus transduction (Yu et al., 2007). At 12-20 days post infection, colonies with human ES cell morphology become visible. The colonies are picked and expanded. The induced pluripotent stem cells making up the colonies are morphologically similar to human ES cells, express various human ES cell markers, and form teratomas having neural tissue, cartilage, and gut epithelium after injection into mice.

[0051] Methods of preparing induced pluripotent stem cells from mouse are also known (Takahashi and Yamanaka, 2006). Induction of iPS cells typically require the expression of or exposure to at least one member from Sox family and at least one member from Oct family. Sox and Oct are thought to be central to the transcriptional regulatory hierarchy that specifies ES cell identity. For example, Sox may be Sox-1, Sox-2, Sox-3,Sox-15, or Sox-18; Oct may be Oct-4. Additional factors may increase the reprogranmiing efficiency, like Nanog, Lin28, Klf4, or c-Myc; specific sets of reprogramming factors may be a set comprising Sox-2, Oct-4, Nanog and, optionally, Lin-28; or comprising Sox-2, Oct4, Kif and, optionally, c-Myc.

[0052] iPS cells, like ES cells, have characteristic antigens that can be identified or confirmed by immunohistochemistry or flow cytometry, using antibodies for SSEA-1, SSEA-3 and SSEA-4 (Developmental Studies Hybridoma Bank, National Institute of Child Health and Human Development, Bethesda Md.), and TRA-1-60 and TRA-1-81 (Andrews et al., 1987). Pluripotency of embryonic stem cells can be confirmed by injecting approximately 0.5-10x106 cells into the rear leg muscles of 8-12 week old male SCID mice. Teratomas develop that demonstrate at least one cell type of each of the three germ layers.

[0053] In certain aspects of the present invention, iPS cells are made from reprogramming somatic cells using reprogramming factors comprising an Oct family member and a Sox family member, such as Oct4 and Sox2 in combination with Kif or Nanog as described above. The somatic cell for reprogramming may be any somatic cell that can be induced to pluripotency, such as a fibroblast, a keratinocyte, a hematopoietic cell, a mesenchymal cell, a liver cell, a stomach cell, or a ~ cell. In a certain aspect, T cells may also be used as source of somatic cells for reprogramming (see U.S. Application No. 61 / 184,546, incorporated herein by reference).

[0054] Reprogramming factors may be expressed from expression cassettes comprised in one or more vectors, such as an integrating vector or an episomal vector, e.g., an EBY element-based system (see U.S. Application No. 61 / 058, 858, incorporated herein by reference; Yu et al., 2009). In a further aspect, reprogramming proteins could be introduced directly into somatic cells by protein transduction (see U.S. Application No. 61 / 172,079, incorporated herein by reference).

[0055] In certain aspects of the invention, there may also be provided methods of transdifferentiation, i.e., the direct conversion of one somatic cell type into another, e.g., deriving hematopoietic precursor cells or hematopoietic cells from non-hematopoietic somatic cells. However, human somatic cells may be limited in supply, especially those from living donors. In certain aspects, to provide an unlimited supply of starting cells for programming, somatic cells may be immortalized by introduction of immortalizing genes or proteins, such as hTERT or oncogenes. The immortalization of cells may be reversible (e.g., using removable expression cassettes) or inducible (e.g., using inducible promoters).

[0056] Somatic cells in certain aspects of the invention may be primary cells (nonimmortalized cells), such as those freshly isolated from an animal, or may be derived from a cell line (immortalized cells). The cells may be maintained in cell culture following their isolation from a subject. In certain embodiments, the cells are passaged once or more than once (e.g., between 2-5, 5-10, 10-20, 20-50, 50-100 times, or more) prior to their use in a method of the invention. In some embodiments the cells will have been passaged no more than 1, 2, 5, 10, 20, or 50 times prior to their use in a method of the invention. They may be frozen, thawed, etc.

[0057] The somatic cells used or described herein may be native somatic cells, or engineered somatic cells, i.e., somatic cells which have been genetically altered. Somatic cells of the present invention are typically marmnalian cells, such as, for example, human cells, primate cells or mouse cells. They may be obtained by well-known methods and can be obtained from any organ or tissue containing live somatic cells, e.g., blood, bone marrow, skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other urinary organs, etc.

[0058] Mammalian somatic cells useful in the present invention include, but are not limited to, Sertoli cells, endothelial cells, granulosa cells, neurons, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macro phages, monocytes, mononuclear cells, cardiac muscle cells, and other muscle cells, etc.

[0059] Somatic cells may be partially or completely differentiated. Differentiation is the process by which a less specialized cell becomes a more specialized cell type. Cell differentiation can involve changes in the size, shape, polarity, metabolic activity, gene expression and / or responsiveness to signals of the cell. For example, hematopoietic stem cells differentiate to give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), erythro-megakaryocytic (erythrocytes, megakaryocytes, thrombocytes), and lymphoid lineages 10 (T-cells, B-cells, natural killer (NK) cells). During progression along the path of differentiation, the ultimate fate of a cell becomes more fixed. As described herein, both partially differentiated somatic cells and fully differentiated somatic 15 cells can be programmed as described herein to produce desired cell types such as hematopoietic cells and hematopoietic precursor cells.

[0060] In one embodiment, the present invention is a method to efficiently produce neutrophils, eosinophils, macrophages, osteoclasts, dendritic and Langerhans cells from mammalian pluripotent stem cells, preferably human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs, see, for example, Yu et al. (2007) Science 318:1917- 1920, incorporated by reference, for one method of making iPSCs) through differentiation of the hESCs or iPSCs into lin-CD34+ CD43+CD45+ myeloid-progenitors enriched cells using the described methods. In some embodiments, cells may further differentiate into lin+CD34-CD43-CD45+ progenitors.

[0061] Pluripotent stem cells hold tremendous promise in modeling human development and disease. They can be derived from either the inner cell mass of a human blastocyst, called human embryonic stem cells (hESCs), or via reprogramming of human somatic cells into pluripotent tissue, called induced pluripotent stem cells or iPSCs (Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872 (2007)). Since the initial derivation of pluripotent stem cells (Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147 (1998)), it is speculated both hESCs and iPSCs can give rise to any cell type in the human body, permitting a genetically tractable, limitless source of human tissue for drug development and cell therapy.

[0062] Hematopoietic progenitors and stem cells (HPSCs) create the blood and immune system for life and sit at a nexus for human disease and disease progression. Atop this hierarchy, hematopoietic stem cells (HSCs) self-renew and are responsible for the lifelong multipotent production of all blood cell fates (Notta, F. et al. Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment. Science 333, 218-221 (2011)), including lymphocytes, myeloid cells, red blood cells, and platelets. HSCs are critical for the normal function of the immune system and the maintenance of hematopoietic homeostasis.

[0063] While the lineage tracing and developmental origin of blood are greatly complicated by the nature of a liquid tissue, evidence from mouse and iPSC studies suggests there are at least 4 independent waves of hemogenic mesoderm (HM) contributing to the blood as the embryo develops (Canu, G. & Ruhrberg, C. First blood: the endothelial origins of hematopoietic progenitors. Angiogenesis 24, 199-211 (2021)): primitive (yolk- sac) hematopoiesis, erythromyeloid progenitor (EMP), lympho-myeloid primed progenitor cell (LMPP), and the long-term hematopoietic stem cell (LT-HSC). The aorta-gonad- mesonephros (AGM) is a mesodermal developmental region where LT-HSCs, capable oflong-term multilineage engraftment (LT-HSCs) in adults first emerge. Human iPSCs permit scientists a window into early human development and are well suited for distinguishing between various waves of blood formation and there has been a tremendous effort to differentiate or reprogram LT-HSCs from either hESCs or iPSCs (Daniel, M.G., Pereira, C.F., Lemischka, I.R. & Moore, K.A. Making a Hematopoietic Stem Cell. Trends Cell Biol 26, 202-214 (2016); Suzuki, N. et al. Generation of engraftable hematopoietic stem cells from induced pluripotent stem cells by way of teratoma formation. Mol Ther 21, 1424-1431 (2013); Elcheva, I. et al. Direct induction of haematoendothelial programs in human pluripotent stem cells by transcriptional regulators. Nat Commun 5, 4372 (2014); Duan, F. et al. Biphasic modulation of insulin signaling enables highly efficient hematopoietic differentiation from human pluripotent stem cells. Stem Cell Res Ther 9, 205 (2018); Sugimura, R. et al. Haematopoietic stem and progenitor cells from human pluripotent stem cells; Nature 545, 432-438 (2017); Zhu, Y. et al. Characterization and generation of human definitive multipotent hematopoietic stem / progenitor cells. Cell Discov 6, 89 (2020); Calvanese, V. et al. Mapping human haematopoietic stem cells from haemogenic endothelium to birth. Nature 604, 534-540 (2022); Sturgeon, C.M., Ditadi, A., Awong, G., Kennedy, M. & Keller, G. Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol 32, 554- 561 (2014)) with most seminal reports indicating great strides towards making an aorta- gonad-mesonephros (AGM) -like HPSC production (Calvanese, V. et al. Mapping human haematopoietic stem cells from haemogenic endothelium to birth. Nature 604, 534-540 (2022); Ng, E.S. et al. Differentiation of human embryonic stem cells to H0XA(+) hemogenic vasculature that resembles the aorta-gonad-mesonephros. Nat Biotechnol 34, 1168-1179 (2016)), with the hallmark of increased lymphocyte production (Kennedy, M. et al. T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures. Cell Rep 2, 1722-1735 (2012)). However, more work is needed in teasing apart cell fate decisions since studies have failed to achieve long-term, multilineage engraftment.

[0064] During human development, LT-HSCs emerge from a hemogenic endothelium by undergoing an endothelial-to-hematopoietic transition (EHT), marked by the expression of Runt-related transcription factor 1 (RUNX1), CD34, and CD45, and the downregulation of the endothelial markers SRY-Box Transcription Factor 17 (SOX17) and CD31. Knock-out studies of the RUNX1 locus in rodents, also called AML1, exhibit normal yolk sac hematopoiesis and embryonic lethality at E12.5 from a lack of fetal liverhematopoiesis (Okuda, T., van Deursen, J., Hiebert, S.W., Grosveld, G. & Downing, J.R. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell 84, 321-330 (1996)). Importantly, studies that focus specifically on the C spliceoform suggest it plays a role in LT-HSC specification, self-renewal, and differentiation (Challen, G.A. & Goodell, M.A. Runxl isoforms show differential expression patterns during hematopoietic development but have similar functional effects in adult hematopoietic stem cells. Exp Hematol 38, 403-416 (2010)), and is required for proper hematopoietic differentiation from iPSCs (Navarro-Montero, O. et al. RUNXlc Regulates Hematopoietic Differentiation of Human Pluripotent Stem Cells Possibly in Cooperation with Proinflammatory Signaling. Stem Cells 35, 2253-2266 (2017)). More efficient protocols allowing observation of cell fate decisions in a discrete and stepwise fashion are necessary to move the field forwards.

[0065] In some embodiments of the invention, we have improved efficiencies of cell fate derivation beyond prior work on the high-efficiency derivation of the nervous system (Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27, 275-280 (2009)) applied to hematopoietic lineages with the goal of making progress towards generating LT-HSCs without using cellular aggregation (embryoid bodies or organoids) or gene delivery, often used for cell fate reprogramming (Chambers, S.M. & Studer, L. Cell fate plug and play: direct reprogramming and induced pluripotency. Cell 145, 827-830 (2011)). Here we report a high efficiency method for generating a HM with all the molecular and many functional hallmarks of a definitive AGM-like wave of hematopoiesis including synchronous EHT, robust expression of TBXT and RUNX1C, single-cell gene expression signature clustering with in vivo counterparts for a population of iPSC derived cells, and robust generation of myeloid and lymphoid cell fates in vitro. The method is amendable to gene editing, even though gene silencing is prevalent within pluripotent stem cell differentiation and is capable of multilineage hematopoiesis persistent for up to 12 weeks in vivo.Generation of lin-CD34+CD43+CD45+ Cell Population

[0066] The present invention is based, in part, on the discovery of a method of producing hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSC). The hPSC can be an inducible pluripotent stem cells (iPSCs), embryonic stem cell, or transdifferentiated somatic cell. The HSCs produced from the methods of the inventioncan differentiate into different hematopoietic lineage cells. The methods of the present invention comprise the following steps:

[0067] A first step is obtaining a cell or a population of human pluripotent stem cells (hPSC), which can be derived from embryonic stem cells, inducible pluripotent stem cells, or transdifferentiated somatic cells, as described above.

[0068] The next step is culturing the cells on day 0 in supplemented serum-free differentiated (SFD) medium (75:25 of IMDM:Ham's F-12, 0.05% BSA, lx B27, 0.5x N2 supplements, IX GlutaMax and IX Penicillin-Streptomycin, 0.5 mM ascorbic acid, 450 pM Monothioglycerol, and 150 pg / mL holo-transferrin). Day 0 represents the day that the differentiation protocol is started, e.g. SFD media is introduced to the population of cells. This allows for potential waiting periods for even distribution of cells, plating of iPSCs, and the like. As such, the cells can be maintained in culture for a period of time prior to introduction of the SFD media. For example, cells may be maintained for up to seven days prior to day 0 introduction of SFD media. Without being bound by theory, this step of introduction of supplemented SFD medium induces hematopoietic and mesoderm differentiation. In some embodiments, the cells can be cultured in supplemented SFD medium for 3, 4, 5, 6, or 7 days. In some embodiments, the cells are cultured in supplemented SFD medium for 3 days. In some embodiments of the invention, BMP4 may be added to the SFD medium in a concentration range from 0.1-500 ng / ml, preferably 1- 100 ng / ml, and even more preferably 5-25 ng / ml. In some embodiments, other BMPs or small molecules that activate ALK1, ALK2, and or ALK3 signaling can be added instead of or in addition to BMP4. In some embodiments, BMP2 or BMP8a may be added instead of or in addition to BMP4 in a concentration range of 1-200 ng / ml. In some embodiments, BMP4, other BMPs and / or small molecules that activate ALK1, ALK2, and or ALK3 signaling may be added to the media on day 0 to day 3. Without being bound by theory, BMP4 and other BMPs or small molecules that activate ALK1, ALK2, and or ALK3 signaling activates SMAD signaling to form mesoderm. In some embodiments, BMP4, other BMPs and / or small molecules that activate ALK1, ALK2, and or ALK3 signaling is a required component of this step of the invention. In some embodiments, bFGF may be added to the media in a concentration range from 1-500 ng / ml, preferably 10-100 ng / ml, and even more preferably 20-50 ng / ml. In some embodiments, other FGFs or MAPk agonists can be added instead of or in addition to bFGF. In some embodiments, bFGF, other FGFs and / or MAPk agonists may be added to the media on day 0 to day 3. Without being bound by theory, bFGF, other FGFs or MAPk agonists aid in survival and patterningto mesoderm. In some embodiments, bFGF, other FGFs and / or MAPk agonists is a required component of this step of the invention. In some embodiments, Y-27632 may be added to the media in a range from 100nM-30pM, preferably lpM-20pM, and even more preferably 5pM-20pM. In some embodiments, Rho kinase inhibitors can be added instead of or in addition to Y-27632. In some embodiments, Y-27632 and / or Rho kinase inhibitors may be added to the media on day 0. Without being bound by theory, Y-27632 and / or Rho kinase inhibitors allow cells to survive as single cells for even distribution in the dish. In some embodiments, CHIR99021 may be added to the media in a range from 0.1-20 pM, preferably 1-10, and even more preferably 5-10 pM. In some embodiments, WNT proteins, other GSK3b inhibitors, and / or small molecules that lead to 0-catenin stabilization, such as Wnt3a, FZM1.8, BIO lithium chloride, CHIR-98014, SB216763, SB415286 can be added instead of or in addition to CHIR99021. In some embodiments, Wnt3a may be added instead of or in addition to CHIR99021 in a concentration range of 1-200 ng / ml, FZM1.8 may be added instead of or in addition to CHIR99021 in a concentration range of 100 nM- 100 pM, BIO may be added instead of or in addition to CHIR99021 in a concentration range of 100 nM-100 pM, lithium chloride may be added instead of or in addition to CHIR99021 in a concentration range of 0.1 mM-20 mM, CHIR-98014 may be added instead of or in addition to CHIR99021 in a concentration range of 500 nM-50 pM, SB216763 may be added instead of or in addition to CHIR99021 in a concentration range of 500 nM-50 pM, and / or SB415286 may be added instead of or in addition to CHIR99021 in a concentration range of 500 nM-50 pM. In some embodiments, CHIR99021, Wnt3a, FZM1.8, BIO lithium chloride, CHIR-98014, SB216763, and / or SB415286 may be added to the media on days 0 to 2, 1 to 2, or only on day 2. Without being bound by theory, CHIR99021, Wnt3a, FZM1.8, BIO lithium chloride, CHIR-98014, SB216763, and / or SB415286 activates Wnt signaling by inhibiting GSK3b. In some embodiments, CHIR99021, Wnt3a, FZM1.8, BIO lithium chloride, CHIR-98014, SB216763, and / or SB415286 is a required component of this step of the invention. In some embodiments, SB- 431542 may be added to the media in a range from 0.1-20 pM. This was found to improve efficiency. In some embodiments, other means to inhibit SMAD signaling, including LY2109761, SB525334, SB505124, GW788388, LY364947, Galunisertib (LY2157299), and / or RepSox may be added instead of or in addition to SB-431542. In some embodiments, LY2109761 may be added instead of or in addition to SB-431542 in a concentration range of 500 nM-50 pM, SB525334 may be added instead of or in additionto SB-431542 in a concentration range of 500 nM-50 pM, SB505124 may be added instead of or in addition to SB-431542 in a concentration range of 500 nM-50 pM, GW788388 may be added instead of or in addition to SB-431542 in a concentration range of 500 nM- 50 pM, LY364947 may be added instead of or in addition to SB-431542 in a concentration range of 500 nM-50 pM, Galunisertib (LY2157299) may be added instead of or in addition to SB-431542 in a concentration range of 500 nM-50 pM, and / or RepSox may be added instead of or in addition to SB-431542 in a concentration range of 500 nM-50 pM. In some embodiments, SB-431542, LY2109761, SB525334, SB505124, GW788388, LY364947, Galunisertib (LY2157299), and / or RepSox may be added to the media on days 1 to 3, on days 2 and 3, or only on day 3. Without being bound by theory, SB-431542, LY2109761, SB525334, SB505124, GW788388, LY364947, Galunisertib (LY2157299), and / or RepSox inhibit ALK / SMAD signaling. In some embodiments, the SFD media is supplemented with BMP4, bFGF, and CHIR99021, in the amounts and times described above. In some embodiments, the SFD media is supplemented with BMP4, bFGF, CHIR99021, and SB-431542, in the amounts and times described above. In one embodiment, the SFD media is supplemented with 10 pM Y-27632 on day 0; 10 ng / ml BMP4 on days 0, 1, and 2; 25 ng / ml bFGF on days 0, 1, and 2; 8 pM CHIR99021 on days 1 and 2; and 6 pM SB-431542 on day 2. The cells are cultured in this media for up to three days. This step is done under hypoxic conditions, which is at an O2 concentration of less than 10%, preferably 5%, and a CO2 concentration of between 1% and 10%, preferably 5%, at 32-39°C, preferably 37°C.

[0069] The next step is culturing the cells in StemPro-34 medium under hypoxic conditions, which is at an O2 concentration of less than 10%, preferably 5%, and a CO2 concentration of between 1% and 10%, preferably 5%, at 32-39°C, preferably 37°C. Without being bound by theory, this step induces endothelium formation. In some embodiments, the cells can be cultured in StemPro-34 medium under hypoxic conditions up to day 4, 5, 6, 7, 8, or 9. In some embodiments, the cells are cultured in supplemented SFD medium up to day 9. In some embodiments of the invention, bFGF may be added to the media in a range from 1-500 ng / ml, preferably 10-100 ng / ml, and even more preferably 20-50 ng / ml. In some embodiments, other FGFs or MAPk agonists can be added instead of or in addition to bFGF. In some embodiments, bFGF, other FGFs or MAPk agonists may be added to the media on day 3 up to day 14 or longer, such as up to day 15, 16, 17, 18, 19, 20, or 21. In some embodiments, SB-431542 may be added to the media in a rangefrom 0.1-20 pM. In some embodiments, other means to inhibit SMAD signaling, including LY2109761, SB525334, SB505124, GW788388, LY364947, Galunisertib (LY2157299), and / or RepSox may be added instead of or in addition to SB-431542. In some embodiments, LY2109761 may be added instead of or in addition to SB-431542 in a concentration range of 500 nM-50 pM, SB525334 may be added instead of or in addition to SB-431542 in a concentration range of 500 nM-50 pM, SB505124 may be added instead of or in addition to SB-431542 in a concentration range of 500 nM-50 pM, GW788388 may be added instead of or in addition to SB-431542 in a concentration range of 500 nM- 50 pM, LY364947 may be added instead of or in addition to SB-431542 in a concentration range of 500 nM-50 pM, Galunisertib (LY2157299) may be added instead of or in addition to SB-431542 in a concentration range of 500 nM-50 pM, and / or RepSox may be added instead of or in addition to SB-431542 in a concentration range of 500 nM-50 pM. In some embodiments, SB-431542, LY2109761, SB525334, SB505124, GW788388, LY364947, Galunisertib (LY2157299), and / or RepSox may be added to the media on day 3, or from day 3 to day 4 or longer, such as up to day 9. In some embodiments, VEGF may be added to the media in a range from 0.1-500 ng / ml, preferably 10-100 ng / ml, and even more preferably 20-50 ng / ml. In some embodiments, drugs that stimulate angiogenesis such as VEGF-C, angiopoietin-1, 2, 3, and / or 4, KDR / FLT-1 agonists, i / eNOS agonists and / or nitric oxide may be added instead of or in addition to VEGF. In some embodiments, VEGF- C, angiopoietin-1, 2, 3, and / or 4 may be added instead of or in addition to VEGF in a concentration range of 1-200 ng / ml. In some embodiments, VEGF may be added to the media on day 3 up to day 14 or longer, such as up to day 15, 16, 17, 18, 19, 20, or 21. Without being bound by theory, VEGF, VEGF-C, angiopoietin-1, 2, 3, and / or 4, KDR / FLT-1 agonists, i / eNOS agonists and / or nitric oxide promote endothelial cell formation and survival. In some embodiments, an HSC cocktail may be added to the media on day 6 up to day 21. The HSC cocktail can contain one or more of: SCF, IL-6, IL-3, FLT3L, IGF-1, IL-11, and EPO. In some embodiments, the HSC cocktail can contain one or more of: SCF, IL-6, IL-3, FLT3L, IGF-1, and / or IL-11, each in a concentration range of 1-200 ng / ml, and / or EPO in a concentration range of 0.1-20 U / ml. In one embodiment, the HSC cocktail contains 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO.

[0070] The next step is culturing the cells in StemPro-34 medium under non- hypoxic conditions, which is at an O2 concentration of greater than 10% up to 30%,preferably normoxic levels or 15-20%, and a CO2 concentration of between 1% and 10%, preferably 5%, 32-39°C, preferably 37°C. Without being bound by theory, this step induces endothelial-hematopoietic transition. In some embodiments, the cells are cultured in StemPro-34 medium under non-hypoxic conditions following StemPro-34 medium under hypoxic conditions (for example, from day 9) up to day 21 and beyond. In some embodiments, the cells can be cultured in StemPro-34 medium under non-hypoxic conditions up to day 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In some embodiments, the cells are cultured in StemPro-34 medium under non-hypoxic conditions up to day 14. In some embodiments of the invention, bFGF may be added to the media in a range from 1-500 ng / ml, preferably 5-50 ng / ml, and even more preferably 10-25 ng / ml. Other compounds that may be added instead of or in addition to bFGF are described above. In some embodiments, bFGF may be added to the media on day 3 up to day 14 or longer (administered both under hypoxic and non-hypoxic conditions), such as up to day 15, 16, 17, 18, 19, 20, or 21. In some embodiments, VEGF may be added to the media in a range from 0.1-500 ng / ml, preferably 10-100 ng / ml, and even more preferably 20-50 ng / ml. Other compounds that may be added instead of or in addition to VEGF are described above. In some embodiments, VEGF may be added to the media on day 3 up to day 14 or longer, such as up to day 15, 16, 17, 18, 19, 20, or 21. In some embodiments, an HSC cocktail may be added to the media on day 6 up to day 21. The HSC cocktail can contain one or more of: SCF, IL-6, IL-3, FLT3L, IGF-1, IL-11, and EPO. Ranges for HSC cocktail components are described above. In one embodiment, the HSC cocktail contains 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO. In some embodiments, EHT cocktail may be added to the media following StemPro-34 medium under hypoxic conditions (for example, from day 9) up to day 14 and beyond, and can be replaced each day. The EHT cocktail can contain one or more of: BMP4 in a concentration range of 1-200 ng / ml, SHH in a concentration range of 1-200 ng / ml, Angiotensin II in a concentration range of 0.1-100 pg / ml, and / or Losartan potassium in a concentration range of 1 .M-1000 .M. In some embodiments, SAG may be added instead of or in addition to SHH in a concentration range of 1-200 ng / ml, preferably 10 ng / ml. In one embodiment, the EHT cocktail contains 10 ng / ml BMP4, 10 ng / ml SHH, lOug / ml Angiotensin II, and lOOuM Losartan potassium, replaced each day.

[0071] The next step is culturing the cells in StemPro-34 medium under non- hypoxic expansion conditions, which is at an O2 concentration of greater than 10% up to30%, preferably normoxic levels or 15-20%, and a CO2 concentration of between 1% and 10%, preferably 5%, 32-39°C, preferably 37°C. In some embodiments, the cells are cultured in StemPro-34 medium under non-hypoxic expansion conditions with HSC cocktail alone. Ranges for HSC cocktail components are described above. In some embodiments, the cells are cultured in StemPro-34 medium under non-hypoxic expansion conditions without EHT cocktail, VEGF, or bFGF. In some embodiments, the HSC cocktail is replaced every 3 days. The HSC cocktail can contain one or more of: SCF, IL- 6, IL-3, FLT3L, IGF-1, IL-11, and EPO. In one embodiment, the HSC cocktail contains 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO. Following this step, HSCs are produced. In some embodiments, the HSCs express CXCR4 on the cell surface.

[0072] For example, the method of the present invention can include the following steps: (a) obtaining a population of pluripotent stem cells, (b) inducing hematopoietic differentiation by culturing on day 0 in SFD medium, 10 uM Y-27632, 10 ng / ml BMP4 and 25 ng / ml bFGF; culturing for 1-2 days with SFD medium, 10 ng / ml BMP4, 5 ng / ml bFGF, and 8 uM CHIR99021; culturing for 1 day with StemPro34 medium,12.5 ng / ml bFGF, and 25 ng / ml VEGF; culturing for 1-2 day with StemPro34 medium,12.5 ng / ml bFGF, and 25 ng / ml VEGF; culturing for 2-4 days with StemPro34 medium,12.5 ng / ml bFGF, 25 ng / ml VEGF, 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO; culturing for 3-5 days with StemPro34 medium, 12.5 ng / ml bFGF, 12.5 ng / ml VEGF, 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, 2 U / ml EPO, 10 ng / ml BMP4, 10 ng / ml SHH, lOug / ml Angiotensin II, and lOOuM Losartan potassium, replaced each day; culturing for 5-10 days with StemPro34 medium, 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO replaced every 3 days.

[0073] In one embodiment, the methods described above induce hematopoietic differentiation and generate lin-CD34+CD43+CD45+ cells. In some embodiments, hematopoietic cells and precursors of hematopoietic cells produced as part of the invention include cells expressing CXCR4, cells that are CD34+,CD45+, CD90+ and THY1+, cells that are CD38-, Lin-, CD43- or CD73-, cells that are CD45+, CD34+, CD90+, CD38-, and Lin-, cells expressing CD90, cells expressing runxlc, or any combination of the above. Runxl is an essential gene for the onset of hematopoiesis, as deletion of RUNX1 causes embryonic lethality. It has also been suggested Runxlc isoform is more specificallyexpressed at the time of definitive hematopoiesis, while Runxla / b is expressed more broadly (Ng et al. (2016) Nat Biotechnol 34(11):1168-79; Challen et al. (2010) Exp Hematol 38(5):403-16; Sroczynska et al. (2009) Blood 114(26): 5279-89; Bos et al. (2015) Development 142(15):2719-24; Bee et al. (2010) Blood 115(15):3042-50).

[0074] One may also use the above identified invention to create cells of myeloid lineage from iPSCs. For example, one may obtain iPSCs as described in Yu et al. (2007) Science 318: 1917- 1920, and differentiate them into lin-CD34+CD43+CD45+ myeloid progenitors enriched cells. Starting from this point, one can then use the above-described protocol.

[0075] In some embodiments, the present invention provides definitive hematopoiesis and generation of long-term repopulating HSCs. In some embodiments, these long-term repopulating HSCs include cells expressing CXCR4, cells that are CD34+ CD45+, CD90+ and THY1+, cells that are CD38-, Lin-, CD43- or CD73-, cells that are CD45+, CD34+, CD90+, CD38-, and Lin-, cells expressing CD90, cells expressing runxlc, or any combination of the above. Without being bound by theory, the expression of CXCR4 is involved in homing of HSCs and long-term population of HSCs to the bone marrow. In some embodiments, HSCs of the present invention include HSCs generated using methods of the present invention, wherein the HSCs express CXCR4 on the cell surface.

[0076] The present invention has been described above with respect to its preferred embodiments. Other forms of this concept are also intended to be within the scope of the claims.Uses for hematopoietic cells and precursors thereof

[0077] The hematopoietic cells and hematopoietic precursor cells provided by methods and compositions of certain aspects of the invention can be used in a variety of applications. These include but are not limited to transplantation or implantation of the hematopoietic cells and hematopoietic precursor in vivo; screening cytotoxic compounds, carcinogens, mutagens growth / regulatory factors, pharmaceutical compounds, etc., in vitro; elucidating the mechanism of hematological diseases and injuries; studying the mechanism by which drugs and / or growth factors operate; diagnosing and monitoring cancer in a patient; gene therapy; and the production of biologically active products, to name but a few.

[0078] Programming-derived hematopoietic and hematopoietic precursor cells of this invention can be used to screen for factors (such as solvents, small molecule drugs, peptides, and polynucleotides) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of hematopoietic cells provided herein.

[0079] In some applications, stem cells (differentiated or undifferentiated) are used to screen factors that promote maturation of cells along the hematopoietic cell lineage, or promote proliferation and maintenance of such cells in long-term culture. For example, candidate hematopoietic cell maturation factors or growth factors are tested by adding them to stem cells in different wells, and then determining any phenotypic change that results, according to desirable criteria for further culture and use of the cells.

[0080] Particular screening applications of this invention relate to the testing of pharmaceutical compounds in drug research. The reader is referred generally to the standard textbook In vitro Methods in Pharmaceutical Research, Academic Press, 1997, and U.S. Pat. No. 5,030,015). In certain aspects of this invention, cells programmed to the hematopoietic lineage play the role of test cells for standard drug screening and toxicity assays, as have been previously performed on hematopoietic cells and precursors in shortterm culture. Assessment of the activity of candidate pharmaceutical compounds generally involves combining the hematopoietic cells or precursors provided in certain aspects of this invention with the candidate compound, determining any change in the morphology, marker phenotype, or metabolic activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlating the effect of the compound with the observed change. The screening may be done either because the compound is designed to have a pharmacological effect on hematopoietic cells or precursors, or because a compound designed to have effects elsewhere may have unintended effects on hematopoietic cells or precursors. Two or more drugs can be tested in combination (by combining with the cells either simultaneously or sequentially), to detect possible drug-drug interaction effects.

[0081] This invention also provides for the use of hematopoietic cells and hematopoietic precursor cells provided herein to restore a degree of function to a subject needing such therapy, perhaps due to a hematological disease or disorder or an injury. For example, hematopoietic cells and hematopoietic precursor cells derived by methods disclosed herein may be used to treat hematological diseases and disorders such as hemoglobinopathies, anemias, etc. In addition, hematopoietic cells and their precursors may be useful in supplying blood or blood cells (such as, for example, red blood cells,platelets, and neutrophil granulocytes) to subjects in need thereof (such as, for example, subjects in need of a blood transfusion or subjects having a hematological disorder). Such cells may be useful for the treatment of hematopoietic cell deficiencies caused by cellsuppressive therapies, such as chemotherapy.

[0082] To determine the suitability of hematopoietic cells and precursors provided herein for therapeutic applications, the cells can first be tested in a suitable animal model. At one level, cells are assessed for their ability to survive and maintain their phenotype in vivo. Programmed cells provided herein are administered to immunodeficient animals (such as NOG mice, or animals rendered immunodeficient chemically or by irradiation) at a site amenable for further observation, such as under the kidney capsule, into the spleen, into a liver lobule, or into the bone marrow. Tissues are harvested after a period of a few days to several weeks or more, and assessed as to whether starting cell types such as pluripotent stem cells are still present. This can be performed by providing the administered cells with a detectable label (such as green fluorescent protein, or P-galactosidase); or by measuring a constitutive marker specific for the administered human cells. Where programmed cells provided herein are being tested in a rodent model, the presence and phenotype of the administered cells can be assessed by immunohistochemistry or ELISA using human specific antibody, or by RT-PCR analysis using primers and hybridization conditions that cause amplification to be specific for human polynucleotide sequences. Suitable markers for assessing gene expression at the mRNA or protein level are provided elsewhere in this disclosure.

[0083] One of the key strengths of human pluripotent stem cells is having control over the timing and order of human development. This enables researchers to observe and understand aspects of early human development otherwise not observed due to access to primary tissue. Capturing a high efficiency definitive AGM-like hematopoiesis from pluripotent stem cells in vitro has been an elusive endeavor. We have been able to observe and demonstrate four discrete phases of development, including mesoderm specification, endothelium specification, EHT, and HPSC expansion and differentiation on the basis of morphology, flow cytometry marker expression, qRT-PCR, single cell sequencing, and functional analysis in vitro and in vivo.

[0084] Definitive hemogenic endothelium arises from an intraembryonic mesoderm lineage, which is marked by the classical T-box transcription factor TBXT. Moreover, in vitro studies of directed differentiation of iPSCs to an intraembryonic mesodermal lineagehave been isolated using the cell surface markers CXCR4 and KDR / FLK1 (Luff, S.A. et al. Identification of a retinoic acid-dependent haemogenic endothelial progenitor from human pluripotent stem cells. Nat Cell Biol 24, 616-624 (2022)). These double-positive cells have been shown to be retinoic acid dependent in their ability to give rise to definitive HSCs, whereas CXCR4 single-positive cells are retinoic acid-independent and primarily give rise to extraembryonic hematopoietic lineages. However, these studies were done in a 3 -dimensional embryoid body (EB) differentiation format that creates signaling gradients. With the use of our TBXT-EGFP reporter, we have demonstrated an additional layer of information as this gene instructs the formation of the primitive streak during gastrulation where intraembryonic mesoderm is specified. Its activity has not been reported to be observed in the formation of extraembryonic mesoderm (REF). We observe the transcriptional activity of TBXT in both the CXCR4+KDR / FLK1- and CXCR4+KDR / FLK1+ populations. Our data suggest that both these populations can give rise to intraembryonic mesoderm in our directed differentiation protocol.

[0085] In contrast to several other reports where hematopoietic progenitor and stem cells have been derived from pluripotent stem cells, we observe the emergence of a RUNX1C+ hematopoiesis up to a week later, between days 14 and 21. The delay in EHT correlates with the later developmental timing of an emergence of a hemogenic endothelium from the AGM and results in a much higher HP SC potency of one in five sorted cells being a CFU as measured by limiting dilution methylcellulose. The higher efficiencies translate into a robust system for generating mature blood cell fates from the iPSC derived HPSCs. One such indicator is the copious production of myeloid and lymphoid cells in vitro. Monocytes and macrophage production are particularly productive protocols wherein we demonstrated the capability of gene editing within the iPSC derived hematopoietic system, despite iPSCs being notorious for silencing genetic material and only a limited number of promotors function upon differentiation of the cells (Norrman, K. et al. Quantitative comparison of constitutive promoters in human ES cells. PLoS One 5, el2413 (2010)).

[0086] One hallmark of a definitive hematopoiesis from iPSCs is increased and more balanced differentiation of lymphocytes from HPSCs (Kennedy, M. et al. T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures. Cell Rep 2, 1722-1735 (2012)). The ATO and in vivo studies are further evidence our iPSC derived HPSCs are definitive hematopoietic progenitors capable of lymphopoiesis. The in vivo differentiation results areparticularly compelling in that they produce a comparable repertoire of B-, T-, and NK- cells and persist in the NSG-SGM3 animal model upwards of 12 weeks when compared to human CB cells.INCORPORATION BY REFERENCE

[0087] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention. To the extent that any of the definitions or terms provided in the references incorporated by reference differ from the terms and discussion provided herein, the present terms and definitions control.EQUIVALENTS

[0088] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and describe the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof.

[0089] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.ExamplesExample 1: Process for generation of hematopoietic stem cells

[0090] iPSC was added in a 6-well plate, coated with poly-L-Ornithine (PLO; Sigma) at a 1 :7 dilution in PBS, with 1ml in each well and incubated at 37°C for 2 hours. The PLO solution was replaced with Laminin (Sigma) in DMEM / F12 at 1 : 150 dilution, with 1ml in each well and incubated at 37°C for 2 hours.

[0091] On day 0, the iPSC were lifted using TrypLE (Thermo Fisher) and 600,000 cells were seeded per well in 2ml SFD medium (75:25 of IMDM:Ham's F-12, 0.05% BSA, lx B27, 0.5x N2 supplements, IX GlutaMax and IX Penicillin- Streptomycin, 0.5 mM ascorbic acid, 450 pM Monothioglycerol, and 150 pg / mL holo-transferrin (R&D Systems)) + 10 uM Y-27632 + 10 ng / ml BMP4 + 25 ng / ml bFGF.

[0092] On day 1, the media was replaced with SFD medium + 10 ng / ml BMP4 + 25 ng / ml bFGF + 8 uM CHIR99021, adding 2ml in each well. On days 2, 3, 4, and 5, 6pM SB-431542 (TOCRIS) was added into the media in some samples. On day 3, the media was replaced with StemPro34 medium + 12.5 ng / ml bFGF + 25 ng / ml VEGF + 6uM SB 431542, adding 2 ml in each well and was incubated for 24 hours. On day 4, the media was replaced with StemPro34 medium + 12.5 ng / ml bFGF + 25 ng / ml VEGF, adding 2 ml in each well and was incubated for 48 hours. On days 6-8, the media was replaced with StemPro34 medium + 12.5 ng / ml bFGF + 25 ng / ml VEGF + 50 ng / ml SCF + 25 ng / ml IL- 6 + 25 ng / ml IL-3 + 25ng / ml FLT3L + 25 ng / ml IGF-1 + 5 ng / ml IL-11 + 2 U / ml EPO, adding 2 ml in each well. On days 9-13, the media was replaced with StemPro34 medium + 12.5 ng / ml bFGF + 12.5 ng / ml VEGF + 50 ng / ml SCF + 25 ng / ml IL-6 + 25 ng / ml IL-3 + 25ng / ml FLT3L + 25 ng / ml IGF-1 + 5 ng / ml IL-11 + 2 U / ml EPO + 10 ng / ml BMP4 + 10 ng / ml SHH + lOug / ml Angiotensin II + lOOuM Losartan potassium, adding 2 ml in each well, with the media replaced every 2~3 days. On days 14-21, the media was replaced with StemPro34 medium + 50 ng / ml SCF + 25 ng / ml IL-6 + 25 ng / ml IL-3 + 25ng / ml FLT3L + 25 ng / ml IGF-1 + 5 ng / ml IL-11 + 2 U / ml EPO, adding 2 ml in each well, with the media replaced every 3 days. On day 21, the cells were FACS sorted for Lin-(CD45RA, CD 10, CD7, CD3, CD19, CD33, CD66b)CD34+CD45+CD38-CD90+ cells. On days 0-10, the cells were incubated at 37°C, 5% O2 and 5% CO2. On days 11-21, the cells were incubated at 37°C, 20% O2 and 5% CO2.Example 2: Assay for presence of hemogenic endothelium and hematopoietic stem cells

[0093] Using the protocol described in Example 1, cells from culture day 9 and 10 were sequenced using single cell sequencing. Hemogenic endothelium are a subset of endothelial cells capable of differentiating into hematopoietic cells. Hemogenic endothelium are characterized as CD34+ THY1+ CD43- CD73-. FACS plots showing the presence of hemogenic endothelium are shown in Figure 1, both in an earlier protocol, aswell as the current protocol shown in Example 1. Further analysis for hematopoietic stem cells (CD34+ CD45+ CD73-) showed a window of endothelial to hematopoietic transition in iPSC cultures to days 19-21 of differentiation. These results are shown in Figure 2.Example 3: Limiting dilution assay to measure multi-lineage potential of iPSC- derived HSC

[0094] FACS was used to purify iPSC-derived putative HSCs (CD34+ CD45+ CD90+ CD38- Lin-). Cells were loaded into wells at 20, 10, 5, 2, or 1 cell(s) / well, each well loaded with methylcellulose with permissive cytokines. The cells were cultured for 14 days and the colonies were scored for colony forming units. The results are shown in Figure 3. Figure 3 A shows the percent of the wells having each cell type when the wells were loaded with a different number of cells. Depending on the number of cells loaded per well, different fractions of cells, including erythroid burst-forming units (BFU-E), macrophage CFU (CFU-M), granulocyte-macrophage CFU (CFU-GM), eosinophil colony-forming units (CFU-E), granulocyte CFU (CFU-G), and multipotential CFU (CFU- GEMM) formed colonies, as shown in Figure 3B.Example 4: Generation of RunxlC-GFP genetic reporter system

[0095] Runxl is an essential gene for the onset of hematopoiesis, as deletion of RUNX1 causes embryonic lethality. It has also been suggested Runxlc isoform is more specifically expressed at the time of definitive hematopoiesis, while Runxla / b is expressed more broadly (Ng et al. (2016) Nat Biotechnol 34(11): 1168-79; Challen et al. (2010) Exp Hematol 38(5):403-16; Sroczynska et al. (2009) Blood 114(26): 5279-89; Bos et al. (2015) Development 142(15):2719-24; Bee et al. (2010) Blood 115(15):3042-50).

[0096] The purpose of creating a GFP-2A-Runxlc genetic reporter line is to fluorescently label the nascent hematopoietic stem cells (HSC) emerging from hemogenic endothelium to allow for expression analysis of runxlc. This reporter line enabled us to visually determine the efficiency of our HSC differentiation protocol, and provided a straitforward readout.

[0097] The targeting design and vector were constructed by using the following steps. Runxlc N-terminus targeting guide RNA 5’-GCATTTTCAGGAGGAAGCGA-3’ (SEQ ID NO: 1) was cloned into pCas9-Guide vector (ORIGENE) using BamHI / BsmBI. The generation of GFP-2A-Runxlc hiPSC reporter line for labeling of hematopoietic stem cells (HSCs) is shown in Figure 4. The “GFP-2A” sequence was inserted before the ATGstart codon of Runxlc exonl, a “LoxP-PGK-BSD-pA-LoxP” cassette was also inserted in intron 1 for enrichment of correctly targeted human induced pluripotent stem cells (hiPSC) clones. The homology arm flanking the knock-in sequence consists of Ikb upstream and downstream of guide RNA targeting site. 7.5 ug pCas9-Ruxnlc-Guide vector and 7.5 ug of GFP-2A-Runxlc donor vector were transfected into 2xl06iPSCs using Lipofectamine 3000. 48 hours post transfection, 2.5 ug / ml blasticidin was applied to enrich targeted population. Cells were selected for 5-7 days and expanded for cryopreservation. Figure 4A shows a schematic picture showing the strategy to target Runxlc genomic locus. Meanwhile, IxlO6blasticidin-enriched iPSCs were harvested for genomic DNA isolation and PCR genotyping test. Figure 4B shows that the primers described in 4A was used for screening positive colonies after genome editing. After blasticidin selection, a total of 48 single cell clones were picked, expanded and subjected PCR genotyping analysis. 38 clones exhibited positive genotyping band on agarose gel (efficiency = 79%). Figure 4C shows an image of the selected positive clone of GFP-2A-Runxlc hiPSC line.

[0098] The following primers in Table 1 were used for genotyping and sequencing of different regions of targeted Runxlc locus:

[0099] Table 1. Primers used for genotyping

[0100] PCR was performed using PfuUltra II Hotstart PCR Master Mix (Agilent), using lOOng genomic DNA of enriched transfection pool. Purified PCR product sequences were confirmed by Sanger sequencing (Genewiz).

[0101] For single cell cloning, blasticidin resistant iPSCs were dissociated into single cells by TryPLE and seeded at single cell density (-2500 cells per 10-cm dish) in mTeSR media. CloneR (Stem Cell Technologies) was added for the first 4 days to promote survival and growth of single cell clones. A second round of blasticidin selection was applied from day 4-7 to further enrich positively targeted clones. Around Day 8-10, colonies emerged from single cell were picked under a microscope in tissue culture cabinet and transferred to 96-well Matrigel coated plates for continuing culture.

[0102] For passaging colony plates, when colonies in the 96-well plate grew to near confluent, cells were dissociated using ReLeSR (Stem Cell Technologies) and resuspended in mTeSR supplemented with lOuM Y-27632 (TOCRIS). The cell suspension were then split into 3x96-well replicate plates at ratio of 1 :3, 1 :5 and 1 :8, respectively. The 1 :5 plate was again dissociated for cryopreservation a few days later.

[0103] To perform PCR screening, when the cells in the 1 :3 plate grew to full confluent, they were lysed using 50ul / well QuickExtract™ DNA Extraction Solution (Lucigen) according to manufacturer’s instructions. 3ul of DNA extraction solution was used as PCR template with primer set LH-In-F / GFP-R for PCR screening. Selected PCR positive colonies were confirmed by PCR with additional primer sets listed in Step 3 and Sanger sequencing.

[0104] Confirmed GFP-2A-Runxlc hiPSC clones were expanded from the 1 :8 replicate plate for downstream applications. Our data showed that RUNX1C-GFP temporal expression highly overlaps with existing HSC markers CD34 and CD45, but only marks a subpopulation of CD34 / CD45 double positive population (see Figure 6). Runxlc-GFP thus serves as an additional marker to further refine the HSC population for higher purity and efficacy.

[0105] Figure 5 shows a visualization of GFP positive hematopoietic stem cells in hiPSC differentiation: GFP-2A-Runxlc iPSCs (dO, top left panel) were firstly differentiated into endothelium (d9, top right panel), followed by induction of endothelial- hematopoietic transition (EHT) that results in emergence of GFP positive hematopoietic stem cells (dl4, mid panel) from selected regions (dashed box, “blood island”) of GFP negative endothelial layer. At day 17, the production of GFP positive HSCs are no longerrestricted in certain regions, but became more prominent throughout the tissue culture (dl7 bottom panel).

[0106] Figure 6 shows a time course of surface marker expression pattern of GFP- 2A-Runxlc iPSCs during hematopoietic differentiation: (A) Single positive population. (B) Runxlc+CD34+CD45+ putative hematopoietic stem cell population.Example 5: Long-term iPSC cell marker expression assay

[0107] CD34 and GFP-Runxlc expression over time

[0108] LT-iPSC and GFP-Runxlc stably expressed LT-iPSC were differentiated using the protocol of Example 1. Attached cells of D9 and suspension cells from Day 14, 16, 17, 20 and 21 were harvested for FACS analysis. All sample groups for FACS were stained with APC-CD34 and sytox blue (Thermo Fisher). FACS analysis were gated on single cells with negative sytox blue staining. Figure 7 shows HSC CD34 vs GFP-Runxlc expression on days 9 and 14. Figure 8 shows HSC CD34 vs GFP-Runxlc expression on days 16 and 17. Figure 9 shows HSC CD34 vs GFP-Runxlc expression on days 20 and 21. In each figure, from left to right were LT-iPSC, GFP-Runxlc over-expressed iPSC and overlay of both cells. GFP-Runxlc started to show expression on Day 14, and expression increased over the time. From Day 14-17, all GFP-Runxlc positive cells were CD34+. Starting from day 20, GFP-Runxlc cells shifted to CD34-.

[0109] Different cell population expression over time

[0110] Different HSC populations were purified by flow cytometry (FACS) sorting. 5000 HSCs from each population were cultured in 5ml MethoCult™ H4435 Enriched (from STEMCELL Technologies Inc.) in 6-well plates (37°C with 5% CO2). After 21 days of culture, all cells in MethoCult™ were collected and diluted in DMEM / F12. After spin down at 1000g x 5min, cell pellets were repeatedly titrated by Pl 000 pipette and single cell numbers were counted by ViaCell. HSC from LT-iPSC and GFP-Runxlc iPSC were sorted based on the gate strategy described above. On Day 16, 17 and 20, only LT : CD45+ / CD34+ were sorted from LT-iPSC and Runxlc: CD34+ / GFP- and Runxlc: CD34+ / GFP- were sorted. On Day 21, all six populations were sorted for CFU assays, as shown in Figure 10. Figure 10 shows cell population sorting for CFU assays from LT-iPSC and GFP-Runxlc iPSC. All HSC from Runxlc-GFP were all gated on CD45+ cells first. All populations represent CD45+ cells. At early stages, GFP-Runxlc expressed HSC generated similar or lower total CFU cells; however, on Day 21, HSC GFP-Runxlc and CD34+ double positiveHSCs are more robust in generating more cells from CFUs (as shown in Figure 11). In all groups, CD34+ is critical to maintain CFU potential.[OHl] Cell type marker analysisHSC cultured in MethoCult™ medium for 21 days, as described above, were harvested, titrated in single cell suspension, blocked by 1% BSA and FcR receptor blocker, stained by antibodies and FACS analysis was performed to check the expression of all lineage surface markers. Figure 12 shows a CFU panel of common progenitor markers. HSC at day 16 which start showing strong Runxlc expression maintain several common progenitor markers after cultured into CFU. As the HSC become more mature, which shown diminished Runxlc expression in CD34+ cells, cells from CFU show minimal common progenitor markers. Figure 13 shows a CFU panel of lymphoid markers. Although MethoCult™ was designed to expand myeloid cells in vitro, small portion of lymphoid lineage cells are identified in CFU. Include T cell, B cell and NK cells. Day 16 HSC shown to be more potent than Day 21 HSC in generating lymphoid lineage cells. Figure 14 shows a CFU panel of myeloid markers. All stage HSC show robust potential to generating myeloid lineage cells in CFU assay. All myeloid lineage cells except platelets were identified in CFU from CD34+ HSC cells.Example 6: HSPC differentiation

[0112] Directed differentiation of endothelium and HPSCs from iPSCs

[0113] For HPSC differentiation, cells were rendered to single cells using Accutase (ICT) and replated on PLO / Laminin treated tissue culture plates in SFD media (IMDM:Ham’s Fl 2, Bovine Albumin, B27, N2, glutamax, Pen / strep, L-ascorbic acid, monotrioglycerol and holo-transferin) containing Y-27632, lOng / ml BMP4, and 25ng / ml bFGF. On day 1, SFD media was added containing 8uM CHIR99021, lOng / ml BMP4, and 25ng / ml bFGF. CHIR99021 and BMP4 were withdrawn on day 3 and 25ng / ml of VEGF was added. The cells were fed from days 6-8 with StemPro34 (Thermo) containing 12.5 ng / ml bFGF, 25ng / ml VEGF, 50ng / ml SCF, 25ng / ml IL-6, 25ng / ml IL-3, 25ng / ml FLT3L, 5ng / ml IL-11, and 2U / ml EPO, and from days 9-13 with the prior media containing lOng / ml BMP4, lOng / ml SHH, lOug / ml Angiotensin II, and lOOuM Losartan K. Finally, on days 14-17 the cells were fed with StemPro34 containing SCF, 25ng / ml IL-6, 25ng / ml IL-3, 25ng / ml FLT3L, 5ng / ml IL-11, and 2U / ml EPO.

[0114] Flow cytometry and immunofluorescence and colony forming units

[0115] HPSCs as defined by positive for CD34, CD45, CD90 and negative for CD38 and a lineage cocktail described above were sorted into a 96-well plate containing human methylcellulose in a limiting dilution of 20, 10, 5, 2, or 1 cell. The 96-well plates were placed in a tissue culture incubator at 37 degrees for 14- 21 days and CFC frequency was calculated based on established methods (Ploemacher, R.E., van der Sluijs, J.P., Voerman, J.S. & Brons, N.H. An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood 74, 2755-2763 (1989)).

[0116] Cell line engineering

[0117] The TBXT and RUNX1C EGFP lines were generated by Al stem. The donor vectors consisted of right and left homology arms containing a P2A-H2B-EGFP followed by an excisable puromycin selection cassette. After transfection, cells were then selected with 0.25pg / mL puromycin for 5 days. Subsequently, cells were plated as single cells for clone selection. PCR amplification was used to detect correctly targeted positive clones for expansion. Screening primers can be found in Table 2.Table 2. Screening primers

[0118] HER2 CAR lentiviral particles were purchased from CreativeBio Labs (CAR-CI) consisting of scFv- CD28-41BB-CD3£a. HER2 CAR LT iPSC lines were generated by lentiviral transduction. Briefly, LT iPSC cultures were treated with Accutase to obtain a single-cell suspension. Approximately 2x105 cells were plated on Matrigel- coated plates in StemFlex media containing Ipg / mL polybrene. Lentiviral particles werethen added to the cell suspension at a multiplicity of infection of (MOI) (Bergen, V., Lange, M., Peidli, S., Wolf, F.A. & Theis, F.J. Generalizing RNA velocity to transient cell states through dynamical modeling. Nat Biotechnol 38, 1408-1414 (2020)). Cultures were then incubated overnight. To select for cells that received the construct, Ipg / mL puromycin was added to the media for 48 hours. Afterward, surviving clones were treated with Accutase to obtain a single-cell suspension and replated in media containing puromycin for an additional 48 hours. To assess the expression of the HER2 CAR, approximately 1x105 cells were incubated with 2.5 pg of FITC conjugated HER2 (Aero Biosystems HE2-HF256) and analyzed by flow cytometry.

[0119] HPSC single-cell sequencing and analysis

[0120] Single-cell RNA-seq was performed using the either the Chromium 3’ V2 or Chromium 3’ V3 chemistry (10X Genomics). Live cells for each sample were sorted and added to reverse transcription master mix with the appropriate volume of RNAse-free water per manufacturer’s guidelines and encapsulated with gel beads using partitioning oil to generate Gel Bead-in-EMulsions (GEMs). After encapsulation, barcoded mRNAs were extracted from GEMs and cleaned. 11 cycles of cDNA amplification were performed using standard temperature settings according to manufacturer’s recommendations. Completed libraries were pooled and sequenced on Illumina NovaSeq6000 S4 flow cells using the sequencing cycle specifications of 26:8:0:98 (Readl :i7 index:i5 index:Read2) for Chromium 3’ V2 or 28:8:0:91 for Chromium 3’ V3. Sample-specific FASTQ files were generated from raw Illumina BCL files using bcl2fastq.

[0121] FASTQ reads were aligned against human reference genome GRCh38-3.0.0 (Cellranger software v3.0.2, 10X Genomics). Raw count matrices of barcoded, mapped reads were then further filtered using a multistep process to get rid of empty droplets and outlier transcriptomes. High-quality transcriptomes were then defined as cell barcodes having >1000 features detected, <10% fraction of mitochondrial RNA reads and a sequence saturation > 40%. The predicted cell cycle phase for each cell was also determined using the machine learning-based approach described by Scialdone et al (Scialdone, A. et al. Computational assignment of cell-cycle stage from single-cell transcriptome data. Methods 85, 54-61 (2015)). All libraries were normalized using the procedure outlined in Ellwanger, et al (Ellwanger, D.C. et al. Prior activation state shapes the microglia response to antihuman TREM2 in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci U S A 118 (2021)). The cell-based size factors were normalized between libraries based on theratio of average UMI counts between libraries using the R package batchelor. The resulting UMI count matrix was log2 -transformed and used for downstream analyses.

[0122] To quantitatively find the gene expression features that have significantly high variance between cells, we used the approach implemented in Ellwanger, et al (Ellwanger, D.C. et al. Prior activation state shapes the microglia response to antihuman TREM2 in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci U S A 118 (2021)). Only genes with expression in >3% of cells were considered. The variance of each gene was fitted as a function of mean expression. Genes were tested for significance using the R package scran by modeling the residuals with an F-distribution, and genes with a p-value < 0.001 and a minimum average expression .1 were considered significant. To ensure that informative variable features were not being excluded, variable feature identification, dimension reduction, and clustering were performed using genes with minimum average expression >0.1 and no novel cell clusters were identified.

[0123] Data dimensionality was reduced using Diffusion Maps (Coifman & Lafon, 2006), since this algorithm can resolve both linear and non-linear substructures across cell states in a deterministic manner. Using the plotExplanatoryVariables function in the R package scater (version 1.0.4), library batch and chemistryspecific (i.e. 10X 3’ V2 versus 10X 3’ V3) variations in cellular expression were detected and determined to be the major confounding variables masking biological variation. Therefore, these factors were regressed out during dimension reduction to increase the signal-to-noise ratio of the spectral embedding. After factor analysis using a scree plot to determine the optimal number of diffusion components to describe variance within the dataset, we used the first 45 components to compute a two-dimensional uniform manifold approximation and projection of single cells.

[0124] To assign clusters (cell states), a shared Nearest Neighbor (SNN) graph was first constructed of the top 45 diffusion components using the R package Seurat (Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36, 411-420 (2018)). Subsequently, the Louvain algorithm implemented in Seurat was used to cluster cells.

[0125] To understand the differentiation dynamics of the dataset, RNA velocity was calculated using scvelo package in python with dynamic modeling and a minimum mass of 3.

[0126] Macrophage differentiation, polarization, MSP, and phagocytosis assay

[0127] The monocytic lineage differentiation protocol followed a previously established hematopoietic differentiation protocol (Yanagimachi, M.D. et al. Robust and highly-efficient differentiation of functional monocytic cells from human pluripotent stem cells under serum- and feeder cell-free conditions. PLoS One 8, e59243 (2013)). The protocol consists of 5 sequential steps by which mature macrophages are differentiated from CD14+ monocytes. In brief, monocytes were cultured in X-vivo 15 medium with XVIVO 15 with 2 mM glutamax, 55 uM b-mercaptoethanol, lx B27, lx N2 and lOOng / ml hMCSF for 7 days, change medium at day 3. For definitive iPSC derived macrophage from HPSCs the same culture medium as monocytes protocol described as above, change medium every 3-4 days and culture for 2 weeks. Macrophages were polarized to Ml phenotype by the culture medium with 25ng / ml IFNg and lOng / ml LPS for 48hrs. Polarized to M2 phenotype by culture medium with 40ng / ml IL4. All MSD experiments were performed using cell supernatant one the Meso Sector S 600MM according to the manufacturer’s protocols.

[0128] The phagocytosis assay is based on a prior study32, and the following equation was used: % specific lysis = [(Sample signal - Tumor alone signal) / (Background signal - Tumor alone signal)] x 100.

[0129] T-cell differentiation

[0130] The T-cell differentiation follows a recently published study from Montel- Hagen, et al. (Montel -Hagen, A. et al. Organoid-Induced Differentiation of Conventional T Cells from Human Pluripotent Stem Cells. Cell Stem Cell 24, 376-389 e378 (2019)). In brief, 5x105 MS5-hDLLl cells were harvested using trypsin and combined with 5x103 of either the iPSC derived definitive HPSCs or CD34 positive human CB cells and in base hematopoietic medium containing EGM2, lOuM Y-27632, and lOuM SB-431542 and centrifuged at 300g. The cell pellet was resuspended in 6 ul fresh hematopoietic induction medium and plated on a 0.4um Millicell trans-well insert. The insert was placed in a well of a 6-well plate containing 1ml of hematopoietic induction medium and media was changed every 2-3 days for 1 week then replaced with media containing hematopoietic cytokines rhTPO 5 ng / ml, rhFLT3L 5 ng / ml, and rhSCF 50 ng / ml. At week 2, T-cell differentiation was induced by changing to RB27 media (RPMI 1640, 4% B27 supplement, 30 iM L-ascorbic acid, 1% Glutamax, 10 ng / ml rhSCF, 5 ng / ml rhFLT3L and 5 ng / ml rhlL- 7.

[0131] Animals, LT-HSC engraftment, and analysis

[0132] Deidentified umbilical cord blood (UCB) CD34+ cells were purchased from Lonza. iPSC derived HSCs were obtained from cultures at day 22 of differentiation and sorted based on CD34+ by Miltenyi magnetic activated cell sorting (MACS). Four- to five- week-old NSG-SGM3 (NSGS) mice were irradiated at a single dose of 1 Gy then conditioned for 4-6 hours. Mice were anesthetized with isoflurane and approximately 3x104 UCB CD34+ and 1x106 iPSC derived cells in 100pL of PBS were delivered via the retroorbital sinus using a 27g insulin needle. Peripheral blood was obtained by terminal cardiac puncture. Blood was then lysed using ACK lysis buffer prior to staining for flow cytometric analysis. Staining cocktail consisted of BV421 CD3, PE CDl lc, BUV496 CD14, FITC CD20, APC-R700 CD33, BUV805- CD56, PE-Cy7 human CD45, and APC murine CD45. Cell viability was determined by 7-AAD.

[0133] HSPCs undergo EHT and emerge between days 14 and 21 in vitro

[0134] The directed differentiation was divided into four discrete stages based on the natural development of the AGM (Figure 15 A, B): mesoderm specification, endothelium specification, EHT, and HPSC expansion and differentiation. By day 9 of cultures, endothelium formation was observed based on morphology and the expression of CD31 (not shown). Between days 14 and 21, phase bright free-floating cells began to emerge indicating the transition of EHT and hematopoiesis (Figure 15B). The harvested suspension cells when examined by flow cytometry were found by sequential gating to be 77% Lin-, 49% CD34 / CD45 positive, 8% CD38 / CD45RA negative, and 80% CD90 positive. A heterozygous iPSC reporter line engineered to express eGFP from the distal RUNX1C promoter was engineered to quantify the emergence of a definitive wave of hematopoiesis using the same method. The emergence of eGFP positive cells were observed in blood islands between days 14 and 21, corresponding with EHT. Upon further examination by flow cytometry, within the CD34 / CD45 positive fraction, nearly all cells were eGFP positive indicating a robust production of definitive wave of HPSCs.

[0135] A limiting dilution methylcellulose colony forming unit (CFU) assay was performed to serve as the gold standard in vitro functional test of HPSCs and from 1 to 20 individual CD34 / 45 / 90 positive, CD38 / Lin negative cells were sorted into a 96-well plate. CFU-granulocyte, erythrocyte, monocyte, and megakaryocyte (CFU-GEMM) colonies could be observed in wells containing as few as two sorted cells (Figure 15F) and an overall colony forming frequency was found to be one in five cells having CFU potential within this sorted fraction (Figure 15G) indicating a high prevalence of HPSCs in the cultures.

[0136] EHT occurs via a TBXT-positive mesodermal intermediate

[0137] A qRT-PCR time course was performed to define the timing and stages of development in the dish to better understand the transition from mesoderm to endothelium. Markers for each stage were observed in sequential time windows corresponding to the morphological changes observed: mesoderm (days 2-4), endothelium and arterial (days 4- 9), and AGM markers (days 4-14). According to a recent study, six reprogramming factors are sufficient to induce an HSC-like state9, perhaps indicating their role in supporting the HSC state by a transcription factor network. Of the six factors, we detected expression of ERG, HOXA5 / 9 / 10, and SPI1, but no LCOR expression was observed.

[0138] Moreover, the T-box transcription factor T (TBXT) is required for normal mesoderm and blood development in rodents20 and its expression appears at gastrulation, and is expressed in and required for posterior and axial mesoderm (Showell, C., Binder, O. & Conlon, F.L. T-box genes in early embryogenesis. Dev Dyn 229, 201-218 (2004)). Additionally, recent reports have shown that extra- versus intraembryonic mesodermal lineages from iPSC differentiations can be further discriminated the expression the cell surface markers CXCR4 and KDR / FLK122, whereas single positive CXCR4 cells give rise to extra-embryonic mesodermal lineages and those cells expressing both CXCR4 and KDR / FLK1 are indicative of intra-embryonic mesoderm.

[0139] We next sought to further assess the developmental origin of our iPSC derived HSCs by examining the expression for the cell surface markers KDR / FLK1 and CXCR4, which have been shown to distinguish between intra- and extra-embryonic mesodermal lineages (Luff, S.A. et al. Identification of a retinoic acid-dependent haemogenic endothelial progenitor from human pluripotent stem cells. Nat Cell Biol 24, 616-624 (2022)). As definitive hematopoiesis arises from an intra-embryonic mesodermal lineage, we examined whether the mesodermal cells at day 3 of differentiation express both these markers (Figure 16B). We find that approximately 40% of cells have coexpression for CRXR4 and KDR / FLK, suggesting that most cells have the potential to give rise to definitive mesodermal lineages.

[0140] To further confirm our observations, we sought to generate a reporter iPSC line for TBXT to assess whether the expression of CXCR4 and KDR / FLK 1 do indeed specify intra-embryonic mesoderm. To do so, we used genetic engineering to introduce a H2B-EGFP construct at the most distal polyadenylation signal site (PAS), immediately following exon 8 of the endogenous TBXT locus (Figure 21C). We then utilized this iPSC line to measure the expression of this lineage priming transcription factor within theCXCR4 and KDR / FLK1 populations. First, we ensured that the engineered line was able to faithfully differentiate to mesoderm by collecting cells on day 3 post differentiation. We then measured the emergence of intra-embryonic mesoderm by flow cytometry. We found that approximately 33.7% of cells were double positive for CXCR4 and KDR / FLK1 (Figure 16C). These results were comparable to the parental from which this line was generated from. Moreover, almost all the cells during this time point of differentiation express EGFP, suggesting TBXT transcriptional activity was present in all four mesodermal populations (Figure 16D). This is further confirmed by overlaying the EGFP intensity onto the four different mesodermal populations that are present on day 3 of differentiation (Figure 16E). Quantification of the TBXT-EGFP mean fluorescent intensity (MFI) revealed that the FLK1-CXCR4- population contained the least amount of TBXT reporter activity, whereas the CXCR4+FLK1+ population was amongst the highest (Figure 16F). Taken together, these data suggest that the use of CXCR4 and FLK1 / KDR does not necessarily discriminate between intra- and extra-embryonic mesoderm as we observe TBXT reporter activity in four populations. Moreover, these data provide further evidence that our differentiation protocol faithfully directs iPSC differentiation cultures to an intra- embryonic mesodermal lineage, thus producing definitive iPSC derived HSCs.

[0141] Single-cell sequencing reveals a population of iPSC derived HSPCs with striking similarities to bone fide hematopoietic stem cells

[0142] To further validate that our iPSC derived HSCs reflect the transcriptional program of those found in humans, we utilized single-cell RNA (scRNA) sequencing to compare the transcriptomes of the various stages of differentiation and the trajectory towards that of bone fide HSC from fetal liver (FL), umbilical cord blood (UCB), and bone marrow (BM). To do so, we sorted cells during specific differentiation time points based on adherence and cell surface markers (Figure 17A). Endothelial cells were isolated as adherent cell types based on the marker CD31, whereas AGM-like cells were identified as CD34+CD45-. Moreover, Pre-HSC populations were characterized as still adherent, but expressing the cell surface markers CD34 and CD45. Lastly, HSCs were identified by CD34+CD45+CD90+ and the more differentiated multipotent progenitors (MPP) by the lack of CD90 expression (CD34+CD45+CD90-). To further aid in the proper calculation of developmental trajectories, CD34-CD45+ cells were included in the experiment to represent those cells that have undergone additional differentiation steps toward terminal cell types of the blood.

[0143] After data integration and clustering, we find that these isolated cell types represent distinct stages of differentiation (Figure 17B). Both endothelial and AGM-like cells cluster independently from Pre-HSCs and HSC populations, suggesting that a developmental transition has occurred in between. This is most likely characterized by the fact that hemogenic endothelial cells undergo an endothelial-to-hematopoietic transition (EHT) from an adherent cell type to suspension (ref). Furthermore, iPSC derived pre-HSCs and HSCs cluster closely together, suggesting a shared transcriptional profile, whereas the MPP population and blood cells cluster away from HSCs. Interestingly, bone fide HSCs from BM, UCB, and FL all cluster together and in approximately the same dimensions as our iPSC derived HSCs, suggesting that our iPSC derived HSCs are transcriptionally related to bone fide HSCs (Figure 17C).

[0144] To ascertain the shared transcriptional profiles of iPSC derived HSCs from that of bone-fide HSCs from human tissues, we looked at cell-type specific genes curated from the literature (Figure 17D). HSCspecific genes, such as GATA2, MIF, DDX21, and CDK6, are expressed solely in both the bone fide HSC populations and are gradually upregulated as iPSC differentiation cultures commit to the HSC fate. These genes show low expression or no expression at all in the endothelial or AGM-like clusters. On the contrary, these clusters have high expression of endothelial-specific genes, such as PECAM1, ID3, and Soxl7. Moreover, MPP and blood cell populations begin to downregulate genes enriched in bone fide and iPSC derived HSCs, while upregulating lineage-specific genes, such as GPX1, LYZ, CD14, and CD3D. To confirm that these gene sets are enriched within the given cell population, we utilized Seurat’s module to score sets of genes (Figure 17E). This analysis reveals enrichment of HSCs genes in both the bone fide and iPSC derived HSCs with no expression of endothelial genes. Additionally, endothelial gene sets were only enriched in the endothelial and AGM-like clusters. This is further exemplified by overlaying celltype specific genes onto the UMAP clusters (Figure 17D). We find that PECAM1, KDR, CDH5, and SOX17 are strongly enriched in clusters that we have annotated as endothelial or AGM-like hemogenic endothelium. As SOX17 has been shown to mark the emergence of hemogenic endothelial (HE)23 and in the process, regulate the HOXA locus24, these data provide further evidence that our differentiation protocol faithfully directs iPSCs towards a definitive HSC fate that is intra- embryonic through a HE intermediate. Additionally, CD34 expression was found in all clusters, however, only pre-HSC, HSC, MPP, and blood cells clusters express PTPRC (CD45), most likely owing to the fact that an EHT event has occurred where nascent HSCsbegin to bud off the adherent HE. Moreover, H0XA9 expression has been shown to be indicative of a definitive HSC program (Calvanese, V. et al. Mapping human haematopoietic stem cells from haemogenic endothelium to birth. Nature 604, 534-540 (2022)). We find that H0XA9 is expressed early on in differentiation in the HE and AGM- like clusters, and its expression is sustained in the HSC clusters. In contrast, HSC-specific genes, such as MYB and MYC, are only expressed in the pre-HSC clusters with no detectable expression in HE and AGM-like clusters. Thus far, gene expression patterns suggest that after mesoderm induction of iPSCs, cells undergo stage-specific developmental transitions to a HE population through an AGM-like stage to become definitive HSCs.

[0145] To further confirm our hypothesis, we performed RNA velocity analysis using scVelo25 of our datasets to elucidate whether our directed differentiation faithfully recapitulates our inferred developmental trajectories (Figure 17G). This analysis not only considers gene expression, but it uses the kinetics of splicing patterns to draw developmental trajectories, therefore is a more reliable method for understanding our in vitro developmental processes. We find that trajectories drawn begin at the endothelial stage and feed into the AGM-like hemogenic endothelial. Moreover, the trajectories also suggest that the AGM-like hemogenic endothelial cells give rise to both the adherent pre- HSC, HSC, MPP, and blood cell clusters, although not directly to the pre-HSC cluster. However, there is a direct trajectory from the pre-HSC cluster to that of the HSC and its more differentiated progeny. Interestingly, the trajectories drawn from the iPSC derived pre-HSC cluster also include inferred trajectories toward bone-fide HSCs.

[0146] Taken together, these data suggest that our directed differentiation protocol faithfully recapitulates the developmental steps in vitro that are required to direct iPSCs to a definitive HSC through a definitive AGM-like hemogenic endothelium.

[0147] HSPCs efficiently generate myeloid cells including polarized macrophage

[0148] Evidence indicates spatial, phenotypical, and functional differences in the successive contributions of myeloid cells originating from primitive through definitive HM (Bergen, V., Lange, M., Peidli, S., Wolf, F.A. & Theis, F.J. Generalizing RNA velocity to transient cell states through dynamical modeling. Nat Biotechnol 38, 1408-1414 (2020)). Human macrophages are extremely plastic in vitro, thus capable of polarization to an Ml or M2 phenotype upon cytokine exposure (Vogel, D.Y. et al. Human macrophage polarization in vitro: maturation and activation methods compared. Immunobiology 219, 695-703 (2014)). While these states may not exactly represent in vivo counterparts, themodel can be extremely useful to examine pro- and anti-inflammatory programs in macrophages for disease modeling. Macrophages were subjected to in vitro polarization from an MO state to either an Ml or M2 state to further tease out differences between primitive and definitive macrophages. We sought to recapitulate these molecular differences between primitive iPSC derived hematopoiesis protocol28 with macrophage generated from the AGM-like definitive HPSCs and their ability to make and polarize macrophage in vitro in comparison to primary human monocyte derived macrophage (Figure 18 A).

[0149] All three populations of macrophage were examined by flow cytometry marker expression of CD 16, CD86, HLA-DR, CD206, TLR2, and TLR4 and striking similarities were observed between them apart by the lower expression of HLA-DR on the iPSC derived macrophage (Figure 18B). PBMC and definitive iPSC derived monocytes had similar flow cytometry profiles when cultured in vitro (Figure 18C). Cytokine profiling was conducted using meso-scale discovery electrochemiluminescence (MSD) to demonstrate both primitive and definitive iPSC derived macrophage upregulated either the proinflammatory cytokines IL-6, IL-8, and TNFa, or the anti-inflammatory cytokine IL- 10 in response to Ml and M2 polarization, respectively (Figure 18E).

[0150] HSPCs can maintain gene edits, including the delivery of a functional HER2 chimeric antigen receptor

[0151] One strength of human iPSCs is that they are amendable to genome engineering and subclones can be created using lentiviral gene delivery to generate cell lines. Human iPSC were transduced using a commercially available CAR consisting of a Trastuzumab derived scFv-CD28-41BB-CD3^ targeting the breast cancer antigen erb-b2 receptor tyrosine kinase 2 (ERBB2 / HER2)29 to evaluate genome silencing during the differentiation to definitive HPSCs, a technical hurdle often encountered during the process of iPSC directed differentiation (Figure 19A). To overcome these hurdles, we chose a lentiviral vector that drives the expression of the HER2 CAR a strong promoter that is not prone to silencing, such as EFl A (Norrman, K. et al. Quantitative comparison of constitutive promoters in human ES cells. PLoS One 5, el2413 (2010)). Additionally, we chose a high multiply infection (MOI) of 25 to ensure enough integrations of the vector into the iPSCs to overcome any heterochromatinization that may occur during differentiation. First, we evaluated the expression of the CAR on the cell surface of iPSCs by incubating the cells with a FITCconjugated HER2 antigen consisting of amino acids threonine 23- threonine 652. Flow cytometric analysis of HER2 antigen binding to CAR-transduced iPSC revealed that most cells express the CAR on the cell surface (Figure 19B). To rule out nonspecific binding of the antigen, untransduced cells were incubated with the HER2 FITC-conjugated antigen. There was no discernable FITC signal detected compared to unstained CAR iPSC lines. To assess whether the expression of the HER2 CAR is sustained after differentiation, we subjected the HER2 CAR iPSC lines toward a definitive HSC using our directed differentiation protocol. We find that robust expression of the HER2 CAR is found on HSPCs derived from our HER2 CAR iPSC lines (Figure 19C). However, we find that there is a drop in differentiation efficiency where the percentage of untransduced LT iPSC HSPCs yield 28.7% CD34+CD45+ compared to the 16.0% yield from the HER2 CAR iPSC lines.

[0152] To test the functionality of these HER2 CAR HSPCs, we directed their differentiation toward macrophages and assessed their ability to phagocytose their target in vitro. To do so, we co-cultured SKOV3 cells that have high expression of HER2 with HER2 CAR macrophages. Macrophages were labeled with a blue CMAC cell tracker dye, whereas SKOV3 cancer cells were labeled with CMPTX. Cells were then mixed at an effector to target (E:T) of 10: 1, where the effort is the CAR macrophage and the target SKOV3 . As the macrophages begin to engulf their target, they become positive for CMPTX as the cancer cell is internalized. Analysis by flow cytometry revealed that iPSC derived macrophages were 23.7% positive for both CMPTX and CMAC blue compared to the 15.2% (Figure 19D). Additionally, we used interferon-gamma as a positive control as it has been shown to increase macrophage phagocytosis (Jorgovanovic, D., Song, M., Wang, L. & Zhang, Y. Roles of IFN-gamma in tumor progression and regression: a review. Biomark Res 8, 49 (2020)). To carefully quantify these phagocytic events in a systematic manner, we calculated the percent specific lysis as discussed here32, by measuring the CMPTX MFI of the co-cultured samples, SKOV3 alone, and macrophages alone. Interestingly, at 24 hours post-co-culture, there is no significant difference between untransduced macrophages compared to CAR macrophages. However, there is a significant increase in phagocytosis at 48 hours post-co-culture comparable to that of interferon-gamma-treated untransduced LT macrophages, suggesting a time-dependent event that does not require macrophage polarization (Figure 19E).

[0153] As the CAR we utilized contains a CD3(^ intracellular signaling domain, we sought to understand how this signaling influences the transcriptional response of iPSC derived macrophages upon engaging the antigen in vitro. First, we polarized our iPSC derived macrophages towards an Ml phenotype and either cultured them alone or withSK0V3 cells. The cultures were then harvested for RNA extracted and submitted for bulk RNA sequencing. We then ran differential expression analysis on the HER2 CAR Ml macrophages cultured with SK0V3 versus HER2 CAR MI macrophages alone. We found 98 upregulated genes with a p-value less than 0.05 and a log2 fold-change greater than one. Gene ontology of biological process reveals that genes upregulated in HER2 CAR Ml macrophages with SKOV3 are involved in cell migration, ERK % signaling, and inflammatory response (Figure 19G). Upregulation of genes involved in cell migration in the presence of SKOV3 suggests that the HER2 CAR directs the macrophage to its target for phagocytosis. Furthermore, we find classic hallmarks of CAR activation in our HER2 CAR macrophages exemplified by upregulated genes involved in the positive regulation of ERK1 and ERK2 cascade, which has been shown to be downstream of CD3(^ TCR signaling (Hwang, J.R., Byeon, Y., Kim, D. & Park, S.G. Recent insights of T cell receptor-mediated signaling pathways for T cell activation and development. Exp Mol Med 52, 750-761 (2020)).

[0154] HSPCs can differentiate into T-cells via an artificial thymic organoid and multilineage blood in vivo

[0155] The iPSC derived definitive HPSCs or CD34 positive human cord blood (CB) cells were aggregated with MS 5 -DLL 1 feeder cells in order to compare the T-cell potential between the two populations by an artificial thymic organoid (ATO) method recently described34 (Figure 20A, B). While the human cord blood cells were three-fold more effective in making CD3 / TCRab positive cells the iPSC derived definitive HPSCs could make T-cells (Figure 20C), and when gating on CD3, the majority of the cells were CD8 single positive, in contrast to the majority of CB cells producing CD4 / 8 double positive T-cells (Figure 20D).

[0156] Robust long-term multilineage blood reconstitution in a conditioned animal model is one functional standard for defining an LT-HSC. We sought to examine the homing and engraftment potential of our iPSC derived definitive HPSCs in comparison to CB cells, known to contain LT-HSCs. iPSC derived definitive HPSCs and CB cells were enriched for CD34 and injected into irradiated immunodeficient NOD triple-transgenic mice expressing human IL3, GM-CSF (CSF2) and SCF (KITLG), called NSGSGM3. The peripheral blood of the NSG-SGM3 mice was examined at 12 weeks post-transplant for both the level of engraftment by human CD45 and multilineage reconstitution using a panel of markers to detect B-cells, T-cells, NK-cells, and monocytes. In four of five CB cell recipients, robust multilineage reconstitution was observed (Figure 20E), and one animallacked robust T-cell contribution. In comparison, two of the five animals transplanted with iPSC derived definitive HPSCs greater than 1% multilineage engraftment was observed (Figure 20F).

Claims

What is Claimed1. A method of producing a hematopoietic precursor cell comprising the steps of: a) obtaining a population of pluripotent stem cells; b) culturing the cells on day 0 in supplemented serum-free differentiated (SFD) medium under a first hypoxic condition; c) culturing the cells in StemPro-34 medium under a second hypoxic condition; d) culturing the cells in StemPro-34 medium under non-hypoxic conditions; and e) culturing the cells in StemPro-34 medium under non-hypoxic expansion conditions; and f) collect population of hematopoietic precursor cells.

2. The method of claim 1, wherein the pluripotent stem cells are human pluripotent stem cells.

3. The method of claim 1 , wherein the supplemented SFD medium is supplemented with one or more of: BMP4, bFGF, Y-27632, CHIR99021, and SB-431542 added to the SFD medium.

4. The method of claim 3, wherein the BMP4 is at a concentration range of 5-25 ng / ml and added to the medium on days 0, 1, and 2; the bFGF is at a concentration range of 20- 50 ng / ml and added to the medium on days 0, 1, and 2; the Y-27632 is at a concentration range of 5pM-20pM and added to the medium on day 0; the CHIR99021 is at a concentration range of 5pM-20pM and added to the medium on days 1 and 2; and the SB- 431542 is at a concentration range of 0.1-20 pM and added to the medium on day 2.

5. The method of claim 4, wherein the BMP4 is at a concentration of 10 ng / ml and added to the medium on days 0, 1, and 2; the bFGF is at a concentration of 25 ng / ml and added to the medium on days 0, 1, and 2; the Y-27632 is at a concentration of 10 pM and added to the medium on day 0; the CHIR99021 is at a concentration range of 5pM-20pM and added to the medium on days 1 and 2; and the SB-431542 is at a concentration range of 0.1-20 pM and added to the medium on day 2.

6. The method of claim 1, wherein the StemPro-34 medium under a second hypoxic condition is supplemented with one or more of: bFGF, HSC cocktail, SB-431542, and VEGF added to the StemPro-34 medium under a second hypoxic condition.

7. The method of claim 6, wherein the bFGF is at a concentration range from 20-50 ng / ml and is added to the medium on day 3 up to day 14; the HSC cocktail comprises 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL- 11, and 2 U / ml EPO and is added to the medium on day 6 up to day 21; the SB-431542 is at a concentration range from 0.1-20 pM and is added to the medium on day 3 to day 9; and the VEGF is at a concentration range from 20-50 ng / ml and is added to the medium on day 3 to day 14.

8. The method of claim 7, wherein the bFGF is at a concentration of 12.5 ng / ml and is added to the medium on day 3 to day 9; the HSC cocktail comprises 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO and is added to the medium on day 6 to day 9; the SB-431542 is at a concentration of 6 pM and is added to the medium on day 3; and the VEGF is at a concentration of 25 ng / ml and is added to the medium on day 3 to day 9.

9. The method of claim 1, wherein the StemPro-34 medium under non-hypoxic condition is supplemented with one or more of: bFGF, HSC cocktail, VEGF, and EHT cocktail added to the StemPro-34 medium under non-hypoxic condition.

10. The method of claim 9, wherein the bFGF is at a concentration range from 10-25 ng / ml and is added to the medium on day 3 up to day 14; the HSC cocktail comprises 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11. and 2 U / ml EPO and is added to the medium on day 6 up to day 21; the VEGF is at a concentration range from 20-50 ng / ml and is added to the medium on day 3 to day 14; and the EHT cocktail comprises BMP4, SHH, Angiotensin II, and Losartan potassium and is added to the medium on day 9 to day 14.

11. The method of claim 10, wherein the bFGF is at a concentration of 12.5 ng / ml and is added to the medium on day 9 to day 14; the HSC cocktail comprises 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO and is added to the medium on day 6 up to day 21; the VEGF is at a concentration of 12.5 ng / ml and is added to the medium on day 9 to day 14; and the EHT cocktail comprises BMP4, SHH, Angiotensin II, and Losartan potassium and is added to the medium on day 9 to day 14.

12. The method of claim 1, wherein the StemPro-34 medium under non-hypoxic expansion condition is supplemented with HSC cocktail added to the StemPro-34 medium under non-hypoxic expansion condition.

13. The method of claim 12, wherein the HSC cocktail comprises 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO.

14. The method of claim 1, wherein the first hypoxic condition contains an O2 concentration less than 10%.

15. The method of claim 1, wherein the second hypoxic condition contains an O2 concentration less than 10%.

16. A method of producing a hematopoietic precursor cell from a pluripotent stem cell or transdifferentiation of a somatic cell, comprising culturing the pluripotent stem cell or somatic cell under conditions to generate the hematopoietic precursor cell that can differentiate into different hematopoietic lineage cells, comprising the steps of (a) obtaining a population of pluripotent stem cells, (b) inducing hematopoietic differentiation by culturing on day 0 in SFD medium, 10 uM Y-27632, 10 ng / ml BMP4 and 25 ng / ml bFGF; culturing for 1-2 days with SFD medium, 10 ng / ml BMP4, 5 ng / ml bFGF, and 8 uM CHIR99021; culturing for 1 day with StemPro34 medium, 12.5 ng / ml bFGF, and 25 ng / ml VEGF; culturing for 1-2 day with StemPro34 medium, 12.5 ng / ml bFGF, and 25 ng / ml VEGF; culturing for 2-4 days with StemPro34 medium, 12.5 ng / ml bFGF, 25 ng / ml VEGF, 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO; culturing for 3-5 days with StemPro34 medium, 12.5 ng / ml bFGF, 12.5 ng / ml VEGF, 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, 2 U / ml EPO, 10 ng / ml BMP4, 10 ng / ml SHH, lOug / ml Angiotensin II, and lOOuM Losartan potassium, replaced each day; culturing for 5-10 days with StemPro34 medium, 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO replaced every 3 days.

17. The method of claim 16, wherein the media with StemPro34 medium, 12.5 ng / ml bFGF, and 25 ng / ml VEGF further comprises 6uM SB 431542.

18. The method of claim 16, wherein the media on days 2, 3, 4, or 5 further comprise 6pm SB 431542 (TOCRIS).

19. The method of claim 1, wherein the pluripotent stem cell is capable of homing to bone marrow.

20. The method of claim 19, wherein the hematopoietic precursor cell expresses CXCR4.

21. The method of claim 1, wherein the hematopoietic precursor cell is CD34+, CD45+, CD90+, or THY1+.

22. The method of claim 1, wherein the hematopoietic precursor cell is CD38-, Lin-, CD43- or CD73-.

23. The method of claim 1, wherein the hematopoietic precursor cell is CD45+, CD34+, CD90+, CD38-, and Lin-.

24. The method of claim 1, wherein the hematopoietic precursor cell is CD90+.

25. The method of claim 1, wherein the hematopoietic precursor cell expresses runxlc.

26. A hematopoietic precursor cell produced using any of the methods of claims 1-25.

27. The hematopoietic precursor cell of claim 26, wherein said cell is capable of long term bone marrow engraftment.

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