Hematopoietic progenitor cell production

Abstract

SUMMARY OF THE INVENTION An improved method for generating hematopoietic progenitor cells from pluripotent stem cells, and the generated hematopoietic progenitor cells, are provided. The present invention provides a method for producing hematopoietic progenitor cells, comprising the steps of: a) obtaining a population of pluripotent stem cells; b) culturing the cells under a first hypoxic condition in supplemented serum-free differentiation (SFD) medium on day 0; c) culturing the cells under a second hypoxic condition in StemPro-34 medium; d) culturing the cells under a non-hypoxic condition in StemPro-34 medium; e) culturing the cells under a non-hypoxic expansion condition in StemPro-34 medium; and f) recovering a population of hematopoietic progenitor cells. The hematopoietic progenitor cells express CXCR4 and are capable of homing to and / or engrafting in the bone marrow.

Description

[Technical Field]

[0001] The present invention relates to the production of hematopoietic stem cells with improved properties. [Background technology]

[0002] Hematopoietic cells, or blood cells, are in high demand for clinical applications and experimental use. In clinical settings, hematopoietic stem cells (HSCs) can be used to reconstitute hematopoiesis in patients who have undergone treatments that suppress hematopoiesis, such as anti-cancer treatments, or in patients with inherited hematologic disorders. Furthermore, red blood cells, platelets, and neutrophil granulocytes can be used in transfusions and in the treatment of certain blood disorders. In the laboratory, blood cells can be used for many applications, including drug screening. Summary of the Invention [Problem to be solved by the invention]

[0003] Currently, blood cells for such clinical and experimental applications are obtained from living donors. However, the limited supply of donor blood limits therapeutic applications and drug screening, especially when genetically compatible donors are required. Therefore, 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.

[0004] Myeloid cells are derived from pluripotent hematopoietic stem cells in the bone marrow and consist of granulocytes (neutrophils, eosinophils, and basophils) and cells of the monocyte / macrophage lineage, including dendritic cells (DCs) and osteoclasts. These cells play important roles in innate and adaptive immunity, inflammatory responses, and bone remodeling.

[0005] We have established a human pluripotent stem cell (hPSC) differentiation protocol to generate hematopoietic stem cells (HSCs). Hematopoiesis occurs in two phases during embryonic development—primitive and definitive. Definitive hematopoiesis is characterized by the generation of long-term repopulating HSCs, which have broad potential for cell therapy and disease modeling, and have not previously been obtained from hPSCs. [Means for solving the problem]

[0006] The present invention is based, in part, on the discovery of a method for producing hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs). In some embodiments, the present invention provides a method for producing hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs), the method comprising the steps of: a) obtaining a population of pluripotent stem cells; b) culturing the cells under a first hypoxic condition in a supplemented serum-free differentiation (SFD) medium on day 0; c) culturing the cells under a second hypoxic condition in StemPro-34 medium; d) culturing the cells under non-hypoxic conditions in StemPro-34 medium; and e) culturing the cells under non-hypoxic expansion conditions in StemPro-34 medium; and f) Recovering the population of hematopoietic progenitor cells A method for producing hematopoietic progenitor cells, comprising:

[0007] In one embodiment, a method for producing hematopoietic progenitor cells from pluripotent stem cells or from transdifferentiation of somatic cells comprises culturing pluripotent stem cells or somatic cells under conditions to generate hematopoietic progenitor cells capable of differentiating into various hematopoietic lineage cells, the method comprising the steps of: (a) obtaining a population of pluripotent stem cells; (b) culturing on day 0 in SFD medium, 10 uM Y-27632, 10 ng / ml BMP4, and 25 ng / ml bFGF; for 1-2 days in SFD medium, 10 ng / ml BMP4, 5 ng / ml bFGF, and 8 uM CHIR99021; for 1 day in StemPro-34 medium, 12.5 ng / ml bFGF, and 25 ng / ml VEGF; for 1-2 days in StemPro-34 medium, 12.5 ng / ml bFGF, and 25 ng / ml VEGF; and for 2-4 days in StemPro-34 medium, 12.5 ng / ml Y-27632, 10 ng / ml BMP4, and 25 ng / ml bFGF. Cultured in bFGF, 25ng / ml VEGF, 50ng / ml SCF, 25ng / ml IL-6, 25ng / ml IL-3, 25ng / ml FLT3L, 25ng / ml IGF-1, 5ng / ml IL-11, and 2U / ml EPO; StemPro-34 medium, 12.5ng / ml bFGF, 12.5ng / ml for 3-5 days. VEGF, 50ng / ml SCF, 25ng / ml IL-6, 25ng / ml IL-3, 25ng / ml FLT3L, 25ng / ml IGF-1, 5ng / ml IL-11, 2U / ml EPO, 10ng / ml BMP4, 10ng / ml The steps include culturing the cells in SHH, 10 μg / ml angiotensin II, and 100 μM losartan potassium, changing the medium every day; and inducing hematopoietic differentiation by culturing the cells in StemPro-34 medium, 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO, changing the medium every three days for 5 to 10 days.

[0008] In some embodiments, the present invention is a hematopoietic progenitor cell, such as a hematopoietic stem cell, produced using the above-described method. In some preferred embodiments, the hematopoietic progenitor cell expresses CXCR4 on the cell surface. In some embodiments, the hematopoietic progenitor cell is CD34+, CD45+, CD90+, or THY1+. In some embodiments, the hematopoietic progenitor cell is CD38-, Lin-, CD43-, and CD73-. In some embodiments, the hematopoietic progenitor cell expresses CD90 on the cell surface. In some embodiments, the hematopoietic progenitor cell expresses Runx1c. In some preferred embodiments, the hematopoietic progenitor cell is capable of generating long-term repopulating hematopoietic progenitor cells. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows FACS plots demonstrating the formation of hemogenic endothelial cells from iPSCs using the previous protocol and the protocol shown in Example 1. [Figure 2] 1 shows generation of HSC-like cells from iPSC-derived hemogenic endothelial cells at day 21. [Figure 3A] Figure 3 shows the results of a limiting dilution assay on day 21 of differentiation. Figure 3A shows the percentage of wells containing each cell type when different numbers of cells were placed in the wells. Figure 3B shows the number of colonies of different cell types formed after different numbers of cells were placed. [Figure 3B] Figure 3 shows the results of a limiting dilution assay on day 21 of differentiation. Figure 3A shows the percentage of wells containing each cell type when different numbers of cells were placed in the wells. Figure 3B shows the number of colonies of different cell types formed after different numbers of cells were placed. [Figure 4A]Generation of a GFP-2A-Runx1c hiPSC reporter line for hematopoietic stem cell (HSC) labeling is shown. Figure 4A shows a schematic diagram illustrating the strategy for targeting the Runx1c genomic locus. Runx1c is transcribed with unique exons from a distal promoter. Guide RNAs were designed to specifically target the ATG start codon of the Runx1c transcript for precise genome editing. A GFP-2A sequence was fused to the N-terminus to fluorescently label differentiating Runx1c-positive HSCs. A LoxP-PGK-BSD-pA-LoxP selection cassette was placed in intron 1 to facilitate enrichment of precisely targeted cell populations. PCR primers (see Table 1) were designed to amplify the left junction between the homologous recombination site and the GFP-2A-Runx1c linker sequence. Figure 4B shows the primers described in 4A used to screen positive colonies after genome editing. After blasticidin selection, a total of 48 single-cell clones were picked, expanded, and subjected to PCR genotyping analysis. 38 clones showed positive genotyping bands on agarose gel (efficiency = 79%). Figure 4C shows an image of selected positive clones of the GFP-2A-Runx1c hiPSC line. [Figure 4B]Generation of a GFP-2A-Runx1c hiPSC reporter line for hematopoietic stem cell (HSC) labeling is shown. Figure 4A shows a schematic diagram illustrating the strategy for targeting the Runx1c genomic locus. Runx1c is transcribed with unique exons from a distal promoter. Guide RNAs were designed to specifically target the ATG start codon of the Runx1c transcript for precise genome editing. A GFP-2A sequence was fused to the N-terminus to fluorescently label differentiating Runx1c-positive HSCs. A LoxP-PGK-BSD-pA-LoxP selection cassette was placed in intron 1 to facilitate enrichment of precisely targeted cell populations. PCR primers (see Table 1) were designed to amplify the left junction between the homologous recombination site and the GFP-2A-Runx1c linker sequence. Figure 4B shows the primers described in 4A used to screen positive colonies after genome editing. After blasticidin selection, a total of 48 single-cell clones were picked, expanded, and subjected to PCR genotyping analysis. 38 clones showed positive genotyping bands on agarose gel (efficiency = 79%). Figure 4C shows an image of selected positive clones of the GFP-2A-Runx1c hiPSC line. [Figure 4C]Generation of a GFP-2A-Runx1c hiPSC reporter line for hematopoietic stem cell (HSC) labeling is shown. Figure 4A shows a schematic diagram illustrating the strategy for targeting the Runx1c genomic locus. Runx1c is transcribed with unique exons from a distal promoter. Guide RNAs were designed to specifically target the ATG start codon of the Runx1c transcript for precise genome editing. A GFP-2A sequence was fused to the N-terminus to fluorescently label differentiating Runx1c-positive HSCs. A LoxP-PGK-BSD-pA-LoxP selection cassette was placed in intron 1 to facilitate enrichment of precisely targeted cell populations. PCR primers (see Table 1) were designed to amplify the left junction between the homologous recombination site and the GFP-2A-Runx1c linker sequence. Figure 4B shows the primers described in 4A used to screen positive colonies after genome editing. After blasticidin selection, a total of 48 single-cell clones were picked, expanded, and subjected to PCR genotyping analysis. 38 clones showed positive genotyping bands on agarose gel (efficiency = 79%). Figure 4C shows an image of selected positive clones of the GFP-2A-Runx1c hiPSC line. [Figure 5] GFP-positive HSCs were visualized during differentiation of hiPSCs. GFP-2A-Runx1c iPSCs (day 0, upper left panel) first differentiated into endothelial cells (day 9, upper right panel), and then underwent endothelial-to-hematopoietic transition (EHT). GFP-positive HSCs emerged from selected regions of the GFP-negative endothelial layer (dashed frame, "blood islands") (day 14, middle panel). By day 17, GFP-positive HSCs were no longer restricted to specific regions but became more prominent throughout the tissue culture (day 17, bottom panel). [Figure 6A] Time course of surface marker expression patterns of GFP-2A-Runx1c iPSCs during hematopoietic differentiation. (A) Single-positive population. (B) Runx1c+CD34+CD45+ putative hematopoietic stem cell population. [Figure 6B]Time course of surface marker expression patterns of GFP-2A-Runx1c iPSCs during hematopoietic differentiation. (A) Single-positive population. (B) Runx1c+CD34+CD45+ putative hematopoietic stem cell population. [Figure 7] HSC CD34 versus GFP-Runx1c expression on days 9 and 14 is shown. [Figure 8] HSC CD34 versus GFP-Runx1c expression on days 16 and 17 is shown. [Figure 9] HSC CD34 versus GFP-Runx1c expression on days 20 and 21 is shown. [Figure 10] 1 shows sorting of cell populations for CFU assay from LT-iPSCs and GFP-Runx1c iPSCs. [Figure 11] Total CFU cell counts on days 16, 17, 20, and 21 are shown. [Figure 12] CFU panels of common progenitor markers on days 16, 17, 20, and 21 are shown. [Figure 13] CFU panels of lymphoid progenitor markers on days 16, 17, 20, and 21 are shown. [Figure 14-1] CFU panels of myeloid progenitor markers on days 16, 17, 20, and 21 are shown. [Figure 14-2] CFU panels of myeloid progenitor markers on days 16, 17, 20, and 21 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0010] Recently, pluripotent stem cell lines have been derived from human fibroblasts by inserting specific genes essential for maintaining the pluripotency of hESCs (Yu, J., et al. 2007, Science. 318:1917-1920; Takahashi, K., et al. 2007, Cell. 131:861-872; Park, IH, 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, extensive proliferation, and differentiation into all three germ layers. It is anticipated that iPSC lines generated from patients with various diseases can be used to obtain any type of progenitor or differentiated cell with specific genetic traits at the cellular level, thus providing a unique opportunity to analyze disease pathogenesis in vitro.

[0011] Previously, a system for hematopoietic differentiation of hESCs into hematopoietic cells by coculture with OP9 bone marrow stromal cells was established (Vodyanik, MA, Bork, JA, Thomson, JA, Slukvin, January 1, 2005, Blood. 105:617-626). This system characterized the emergence of two subpopulations of the most primitive pluripotent hematopoietic cells in cocultures of hESCs with OP9, based on their common expression of CD43 and differential expression of CD45. Lin-CD34+CD43+CD45- cells, which have broad lymphoid and myeloid differentiation potential, first emerge in the coculture. Subsequently, lin-CD34+CD43+CD45+ cells, enriched for myeloid progenitors, emerge (Vodyanik, MA, Thomson, JA, Slukvin, January 2, 2006, Blood. 108:2095-2105). The Slukvin laboratory demonstrated that a similar pattern of hematopoietic differentiation was observed when iPSCs were differentiated into blood cells in co-culture with OP9 (Choi, K., et al. 2009, Stem Cells. 27:559-567).

[0012] In certain embodiments of the present invention, methods and compositions are disclosed for providing hematopoietic cells or precursors of hematopoietic cells by forward programming of non-hematopoietic human pluripotent cells, including stem cells, including human embryonic stem cells and induced pluripotent stem cells, or by transdifferentiation of non-hematopoietic somatic cells. Cells are also provided that contain an exogenous expression cassette comprising one or more hematopoietic progenitor cell programming factor genes and / or a reporter expression cassette specific for the identification of hematopoietic cells or hematopoietic progenitor cells. In some embodiments, the cells can be stem cells, including, but not limited to, embryonic stem cells, fetal stem cells, or adult stem cells. In further embodiments, the cells can be any somatic cell.

[0013] Stem cells are cells found in most, if not all, multicellular organisms. They are characterized by their ability to renew themselves through cell division and to differentiate into various specialized cell types. The two main types of mammalian stem cells are embryonic stem cells, found in blastocysts, and adult stem cells, found in adult tissues. In the developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In the adult, stem and progenitor cells act as the body's repair system, replenishing specialized cells and maintaining the normal turnover of regenerative organs such as blood, skin, or intestinal tissue.

[0014] 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 ability to differentiate into all cell types of the body, including hematopoietic cells and hematopoietic progenitor cells. Therefore, these cells appear to potentially provide an unlimited supply of patient-specific functional hematopoietic cells and hematopoietic progenitor cells for both drug development and therapeutic use. Differentiation of human ESCs / iPSCs into hematopoietic cells and hematopoietic progenitor cells in vitro recapitulates normal in vivo development; i.e., they undergo normal sequential developmental stages, including mesodermal differentiation and hematopoietic specification. This sequential developmental process requires the addition of different growth factors at different stages of differentiation. Certain embodiments of the present invention provide fully functional hematopoietic progenitor cells by forward programming from human ESCs / iPSCs or by transdifferentiation from somatic cells, bypassing most, if not all, normal developmental stages, by expressing a combination of transcription factors critical for hematopoietic cell differentiation / function, similar to the generation of iPSCs. This approach can be more time- and cost-effective in generating hematopoietic progenitor cells and hematopoietic cells with functions highly similar, if not identical, to those of human adult hematopoietic cells and hematopoietic cell precursors. Additionally, human ESCs / iPSCs, due to their unlimited proliferation potential, may be advantageous over somatic cells as a starting cell population for hematopoietic progenitor differentiation. Examples of hematopoietic cells and hematopoietic cell precursors produced as part of the present invention include cells that express 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 that express CD90, cells that express Runx1c, or any combination of the above.

[0015] 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 early morula stage embryo. Blastocysts are early-stage embryos, approximately 4-5 days old in humans, and consist of 50-150 cells. ES cells are pluripotent and, during development, give rise to the induction of all three primary germ layers: ectoderm, endoderm, and mesoderm. In other words, ES cells can develop into each of the cell types of the adult body when given sufficient and necessary stimuli for that particular cell type. ES cells do not contribute to the extraembryonic membranes or placenta.

[0016] To date, most studies have used mouse embryonic stem cells (mES) or human embryonic stem cells (hES). While both possess essential stem cell properties, they require very different environments to maintain their undifferentiated state. Mouse ES cells can be grown on a layer of gelatin and require the presence of leukemia inhibitory factor (LIF). Human ES cells can 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.

[0017] Human embryonic stem cells can also be defined by the presence of multiple transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and Sox-2 form a central regulatory network that ensures the repression of genes leading to differentiation and the maintenance of pluripotency (Boyer et al., 2005). Cell surface antigens widely used to identify hES cells include the glycolipids SSEA3 and SSEA4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81.

[0018] 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 anti-human spleen cell antiserum followed by 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 in the presence of fetal bovine serum. After 9-15 days, clumps of cells derived from the inner cell mass can be chemically (i.e., by exposure to trypsin) or mechanically dissociated and replated in fresh medium containing fetal bovine serum and a feeder layer of mouse embryonic fibroblasts. After further expansion, colonies with undifferentiated morphology are selected with a micropipette, mechanically dissociated into clumps, and replated (see U.S. Pat. No. 6,833,269). ES-like morphology is characterized by tightly packed colonies with a clearly high nuclear-to-cytoplasmic ratio and prominent nucleoli. The resulting ES cells can be routinely passaged by brief trypsinization or by selecting individual colonies with a micropipette. In some methods, human ES cells can be grown without serum by culturing them on a fibroblast feeder layer 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 a "conditioned" medium containing basic fibroblast growth factor (Xu et al., 2001). The medium is conditioned by prior co-culturing with fibroblasts.

[0019] Another source of ES cells is established ES cell lines. Various mouse and human ES cell lines are known, and conditions for their growth and proliferation have been defined. For example, the mouse CGR8 cell line was established from the inner cell mass of mouse 129 embryos, and CGR8 cell cultures can be grown without a feeder layer in the presence of LIF. As a further example, human ES cell lines H1, H7, H9, H13, and H14 were established by Thompson et al. In addition, subclones of the H9 line, H9.1 and H9.2, have been developed. It is believed that almost any ES cell line or stem cell line known in the art can be used in connection with the present invention, such as those described in Yu and Thompson (2008) Genes Dev 22(15):1987-97, which is incorporated herein by reference.

[0020] The source of ES cells for use in connection with the present invention can be blastocysts, cells derived from cultures of the inner cell mass of blastocysts, 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 blastocysts, ES cells obtained from cultures of inner cell mass cells, and ES cells obtained from cultures of ES cell lines.

[0021] Induced pluripotent stem (iPS) cells are cells that have the properties of embryonic stem cells but are obtained by reprogramming differentiated somatic cells. Induced pluripotent stem cells have been obtained by various methods. In one method, adult human dermal fibroblasts are transfected with the 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 fibroblast cell line that produces basic fibroblast growth factor (LIF)) in medium supplemented with basic fibroblast growth factor (bFGF). After approximately 25 days, colonies resembling human embryonic stem cell colonies appear in culture. ES cell-like colonies are picked and expanded on feeder cells in the presence of bFGF.

[0022] Based on cell characteristics, the cells of the ES cell-like colonies are induced pluripotent stem cells. Induced pluripotent stem cells are morphologically similar to human ES cells and express various human ES cell markers. Furthermore, induced pluripotent stem cells differentiate appropriately when grown under conditions known to result in the differentiation of human ES cells. For example, induced pluripotent stem cells can differentiate into cells with hematopoietic cell structures and markers. It is believed that almost any iPS cell or cell line can be used in connection with the present invention, including, for example, those described in Yu and Thompson, 2008.

[0023] In another method, lentiviral transduction is used to transfect human fetal or neonatal fibroblasts with four genes: Oct4, Sox2, Nanog, and Lin28 (Yu et al., 2007). Twelve to twenty days after infection, colonies with human ES cell morphology become visible. The colonies are then harvested and expanded. The induced pluripotent stem cells that comprise the colonies are morphologically similar to human ES cells, express various human ES cell markers, and, after injection into mice, form teratomas containing neural tissue, cartilage, and intestinal epithelium.

[0024] Methods for preparing induced pluripotent stem cells from mice are also known (Takahashi and Yamanaka, 2006). Induction of iPS cells typically requires the expression of, or exposure to, at least one member of the Sox family and at least one member of the Oct family. Sox and Oct are thought to be central to the transcriptional regulatory hierarchy that specifies the identity of ES cells. For example, Sox can be Sox-1, Sox-2, Sox-3, Sox-15, or Sox-18, and Oct can be Oct-4. Additional factors such as Nanog, Lin28, Klf4, or c-Myc can enhance reprogramming efficiency. The characteristic set of reprogramming factors can be a set including Sox-2, Oct-4, Nanog, and optionally Lin-28, or a set including Sox-2, Oct4, Klf4, and optionally c-Myc.

[0025] Like ES cells, iPS cells have characteristic antigens that can be identified or confirmed by immunohistochemistry or flow cytometry using antibodies against SSEA-1, SSEA-3, and SSEA-4 (Developmental Studies Hybridoma Bank, National Institute of Child Health and Human Development, Bethesda, Md.), and antibodies against TRA-1-60 and TRA-1-81 (Andrews et al., 1987). The pluripotency of embryonic stem cells was confirmed by injecting approximately 0.5–10 × lO cells into the hindlimb muscles of 8–12-week-old male SCID mice. 6 This can be confirmed by injecting cells into the blastocyst, resulting in teratomas that display at least one cell type from each of the three germ layers.

[0026] In certain embodiments of the present invention, iPS cells are generated from reprogrammed somatic cells using reprogramming factors including Oct family members and Sox family members, e.g., Oct4 and Sox2, in combination with Kif or Nanog, as described above. Somatic cells for reprogramming can be any somatic cells that can be induced to become pluripotent, such as fibroblasts, keratinocytes, hematopoietic cells, mesenchymal cells, liver cells, gastric cells, or T cells. In certain embodiments, T cells can also be used as a source of somatic cells for reprogramming (see U.S. Patent Application No. 61 / 184,546, which is incorporated herein by reference).

[0027] The reprogramming factors can be expressed from expression cassettes contained in one or more vectors, e.g., integrative or episomal vectors, such as the EBY element-based system (see U.S. Patent Application No. 61 / 058,858, which is incorporated herein by reference; see Yu et al., 2009). In a further embodiment, the reprogramming proteins can be directly introduced into somatic cells by protein transduction (see U.S. Patent Application No. 61 / 172,079, which is incorporated herein by reference).

[0028] In certain embodiments of the present invention, methods of transdifferentiation, i.e., the direct conversion of one somatic cell type to another, for example, methods of deriving hematopoietic progenitor cells or hematopoietic cells from non-hematopoietic somatic cells, are also provided. However, human somatic cells, particularly those from living donors, can be in limited supply. In certain embodiments, somatic cells can be immortalized by the introduction of an immortalizing gene or protein, such as hTERT or an oncogene, to provide an unlimited supply of starting cells for programming. Cell immortalization can be reversible (e.g., using a removable expression cassette) or inducible (e.g., using an inducible promoter).

[0029] In certain aspects of the present invention, somatic cells may be primary cells (non-immortalized cells), e.g., freshly isolated from an animal, or may be derived from a cell line (immortalized cells). Cells may be maintained in cell culture after isolation from a subject. In certain embodiments, cells are passaged one or more times (e.g., 2-5 times, 5-10 times, 10-20 times, 20-50 times, 50-100 times, or more) before use in the methods of the present invention. In some embodiments, cells will have been passaged 1, 2, 5, 10, 20, or 50 times or less before use in the methods of the present invention. Cells may be frozen, thawed, etc.

[0030] Somatic cells as used or described herein may be naturally occurring somatic cells or engineered somatic cells, i.e., genetically modified somatic cells. The somatic cells of the present invention are typically mammalian cells, such as human cells, primate cells, or mouse cells. They may be obtained by well-known methods or from any organ or tissue containing living cells, such as blood, bone marrow, skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra, and other urinary organs.

[0031] Mammalian somatic cells useful in the present invention include, but are not limited to, Sertoli cells, endothelial cells, granulosa cells, nerve cells, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B lymphocytes and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, cardiac muscle cells, and other muscle cells.

[0032] Somatic cells can be partially or fully differentiated. Differentiation is the process by which less specialized cells become more specialized cell types. Cell differentiation can change size, shape, polarity, metabolic activity, gene expression, and / or responsiveness to cellular signals. For example, hematopoietic stem cells differentiate to give rise to all blood cell types, including the myeloid lineage (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), the erythroid-megakaryocytic lineage (erythrocytes, megakaryocytes, platelets), and the lymphoid lineage (T cells, B cells, natural killer (NK) cells). As a cell progresses along the differentiation pathway, its ultimate fate becomes more determined. As described herein, both partially and fully differentiated somatic cells can be programmed as described herein to produce desired cell types, such as hematopoietic cells and hematopoietic progenitor cells.

[0033] In one embodiment, the invention is a method for efficiently producing neutrophils, eosinophils, macrophages, osteoclasts, dendritic cells, and Langerhans cells from mammalian pluripotent stem cells, preferably human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs; see, e.g., Yu et al. (2007) Science 318:1917-1920, which is incorporated by reference for one method of generating iPSCs), by differentiation of hESCs or iPSCs into cells enriched for lin-CD34+CD43+CD45+ myeloid progenitor cells using methods described therein. In some embodiments, the cells can be further differentiated into lin+CD34-CD43-CD45+ progenitor cells.

[0034] Generation of lin-CD34+CD43+CD45+ cell population The present invention is based, in part, on the discovery of a method for producing hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs). The hPSCs can be induced pluripotent stem cells (iPSCs), embryonic stem cells, or transdifferentiated somatic cells. The HSCs produced from the methods of the invention can be differentiated into a variety of hematopoietic lineage cells. The methods of the invention include the following steps:

[0035] The first step is to obtain a cell or population of human pluripotent stem cells (hPSCs), which can be derived from embryonic stem cells, induced pluripotent stem cells, or transdifferentiated somatic cells, as described above.

[0036] The next step is culturing the cells in supplemented serum-free differentiation (SFD) medium (75:25 IMDM:Ham's F-12, 0.05% BSA, 1x B27, 0.5x N2 supplement, 1x GlutaMax and 1x penicillin-streptomycin, 0.5 mM ascorbic acid, 450 µM monothioglycerol, and 150 µg / mL holo-transferrin) on day 0. Day 0 is the day the differentiation protocol begins, i.e., the day SFD medium is introduced to the population of cells. This allows for even distribution of cells and a possible waiting period for iPSC seeding, etc. For this reason, the cells can be maintained in culture for a period of time before introducing SFD medium. For example, the cells may be maintained for up to 7 days before introducing SFD medium on day 0. Without being bound by theory, this step of introducing supplemented SFD medium induces hematopoietic and mesodermal differentiation. In some embodiments, cells may be cultured in supplemented SFD medium for 3, 4, 5, 6, or 7 days. In some embodiments, cells are cultured in supplemented SFD medium for 3 days. In some embodiments of the present invention, BMP4 may be added to SFD medium at a concentration ranging from 0.1 to 500 ng / ml, preferably from 1 to 100 ng / ml, and even more preferably from 5 to 25 ng / ml. In some embodiments, other BMPs or small molecules that activate ALK1, ALK2, and / or ALK3 signaling may 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 at a concentration ranging from 1 to 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 medium on days 0 to 3. Without being bound by theory, BMP4 and other BMPs, or small molecules that activate ALK1, ALK2, and / or ALK3 signaling, activate SMAD signaling to form mesoderm, hi some embodiments, BMP4, other BMPs, and / or small molecules that activate ALK1, ALK2, and / or ALK3 signaling are essential components of this step of the present invention.In some embodiments, bFGF may be added to the culture medium at a concentration ranging from 1 to 500 ng / ml, preferably from 10 to 100 ng / ml, and even more preferably from 20 to 50 ng / ml. In some embodiments, other FGFs or MAPk agonists may be added instead of or in addition to bFGF. In some embodiments, bFGF, other FGFs, and / or MAPk agonists may be added to the culture medium on days 0 to 3. Without being bound by theory, bFGF, other FGFs, or MAPk agonists aid in survival and mesoderm patterning. In some embodiments, bFGF, other FGFs, and / or MAPk agonists are essential components of this step of the method. In some embodiments, Y-27632 may be added to the culture medium at a concentration ranging from 100 nM to 30 μM, preferably from 1 μM to 20 μM, and even more preferably from 5 μM to 20 μM. In some embodiments, a Rho kinase inhibitor may be added instead of or in addition to Y-27632. In some embodiments, Y-27632 and / or a Rho kinase inhibitor may be added to the culture medium on day 0. Without being bound by theory, Y-27632 and / or a Rho kinase inhibitor allows cells to distribute evenly within the dish and survive as single cells. In some embodiments, CHIR99021 may be added to the culture medium at a concentration of 0.1-20 μM, preferably 1-10 μM, and even more preferably 5-10 μM. In some embodiments, small molecules that stabilize WNT proteins, other GSK3b inhibitors, and / or β-catenin, such as Wnt3a, FZM1.8, BIO, lithium chloride, CHIR-98014, SB216763, and SB415286, may be added instead of or in addition to CHIR99021.In some embodiments, Wnt3a may be added in place of or in addition to CHIR99021 at a concentration range of 1 to 200 ng / ml, FZM1.8 may be added in place of or in addition to CHIR99021 at a concentration range of 100 nM to 100 μM, BIO may be added in place of or in addition to CHIR99021 at a concentration range of 100 nM to 100 μM, and lithium chloride may be added in place of or in addition to CHIR99021. In some embodiments, CHIR99021, Wnt3a, FZM1.8, BIO, lithium chloride, CHIR-98014, SB216763, and / or SB415286 may be added to the medium on days 0 and 2, on days 1 and 2, or on day 2 only. Without being bound by theory, CHIR99021, Wnt3a, FZM1.8, BIO, lithium chloride, CHIR-98014, SB216763, and / or SB415286 activate Wnt signaling by inhibiting GSK3b. In some embodiments, CHIR99021, Wnt3a, FZM1.8, BIO, lithium chloride, CHIR-98014, SB216763, and / or SB415286 are essential components of this step of the method. In some embodiments, SB-431542 may be added to the medium in the range of 0.1 to 20 μM. This has been found to increase efficiency. In some embodiments, other means of inhibiting SMAD signaling, such as 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 in place of or in addition to SB-431542 at a concentration range of 500 nM to 50 μM, SB525334 may be added in place of or in addition to SB-431542 at a concentration range of 500 nM to 50 μM, SB505124 may be added in place of or in addition to SB-431542 at a concentration range of 500 nM to 50 μM, and GW788388 may be added in place of or in addition to SB-431542. Additionally, LY364947 may be added in the concentration range of 500 nM to 50 μM in place of or in addition to SB-431542, galunisertib (LY2157299) may be added in the concentration range of 500 nM to 50 μM in place of or in addition to SB-431542, and / or RepSox may be added in the concentration range of 500 nM to 50 μM in place of or in addition to SB-431542. In some embodiments, SB-431542, LY2109761, SB525334, SB505124, GW788388, LY364947, galunisertib (LY2157299), and / or RepSox may be added to the medium on days 1 through 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 medium is supplemented with BMP4, bFGF, and CHIR99021 in the amounts and for the times described above. In some embodiments, SFD medium is supplemented with BMP4, bFGF, CHIR99021, and SB-431542 in the amounts and for the times described above. In one embodiment, SFD medium is supplemented with 10 μM 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 μM CHIR99021 on days 1 and 2; and 6 μM SB-431542 on day 2. Cells are cultured in this medium for up to 3 days.This step is carried out under hypoxic conditions with an O2 concentration of less than 10%, preferably 5%, and a CO2 concentration between 1% and 10%, preferably 5%, at 32-39°C, preferably 37°C.

[0037] The next step is culturing the cells in StemPro-34 medium under hypoxic conditions, with an O concentration of less than 10%, preferably 5%, and a CO concentration between 1% and 10%, preferably 5%, at 32-39°C, preferably 37°C. Without being bound by theory, this step induces endothelial formation. In some embodiments, the cells can be cultured in StemPro-34 medium under hypoxic conditions for up to day 4, 5, 6, 7, 8, or 9. In some embodiments, the cells are cultured in supplemented SFD medium for up to day 9. In some embodiments of the present invention, bFGF may be added to the medium in the range of 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 FGF, or MAPk agonists may be added to the culture medium from day 3 to day 14, or for longer periods, such as days 15, 16, 17, 18, 19, 20, or 21. In some embodiments, SB-431542 may be added to the culture medium in the range of 0.1-20 μM. In some embodiments, other means of inhibiting SMAD signaling, such as 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 in place of or in addition to SB-431542 at a concentration range of 500 nM to 50 μM, SB525334 may be added in place of or in addition to SB-431542 at a concentration range of 500 nM to 50 μM, SB505124 may be added in place of or in addition to SB-431542 at a concentration range of 500 nM to 50 μM, and GW788388 may be added in place of or in addition to SB-431542. Additionally, LY364947 may be added in the concentration range of 500 nM to 50 μM in place of or in addition to SB-431542, galunisertib (LY2157299) may be added in the concentration range of 500 nM to 50 μM in place of or in addition to SB-431542, and / or RepSox may be added in the concentration range of 500 nM to 50 μM in place of or in addition to SB-431542. In some embodiments, SB-431542, LY2109761, SB525334, SB505124, GW788388, LY364947, galunisertib (LY2157299), and / or RepSox may be added to the culture medium on day 3, or on days 3-4, or for longer periods, such as up to day 9. In some embodiments, VEGF may be added to the culture medium at a concentration ranging from 0.1 to 500 ng / ml, preferably 10 to 100 ng / ml, and even more preferably 20 to 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 in place of or in addition to VEGF at a concentration ranging from 1 to 200 ng / ml. In some embodiments, VEGF may be added to the medium from day 3 to day 14, or for longer periods, such as 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 culture medium from day 6 to day 21. The HSC cocktail may contain one or more of SCF, IL-6, IL-3, FLT3L, IGF-1, IL-11, and EPO. In some embodiments, the HSC cocktail may contain one or more of SCF, IL-6, IL-3, FLT3L, IGF-1, and / or IL-11, each at a concentration range of 1 to 200 ng / ml, and / or EPO at a concentration range of 0.1 to 20 U / ml. In one embodiment, the HSC cocktail contains 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO.

[0038] The next step is culturing the cells in StemPro-34 medium under non-hypoxic conditions, with an O concentration greater than 10% and up to 30%, preferably at normoxic levels or 15-20%, and a CO concentration between 1% and 10%, preferably 5%, at 32-39°C, preferably 37°C. Without being bound by theory, this step induces an endothelial-to-hematopoietic transition. In some embodiments, cells are cultured in StemPro-34 medium under non-hypoxic conditions (e.g., from day 9) after hypoxic conditions in StemPro-34 medium until day 21 and beyond. In some embodiments, cells can be cultured in StemPro-34 medium under non-hypoxic conditions until day 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In some embodiments, cells are cultured in StemPro-34 medium under non-hypoxic conditions for up to 14 days. In some embodiments of the present invention, bFGF may be added to the medium in the range of 1 to 500 ng / ml, preferably 5 to 50 ng / ml, and even more preferably 10 to 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 medium from day 3 to day 14, or for longer periods (performed under both hypoxic and non-hypoxic conditions), for example, up to day 15, 16, 17, 18, 19, 20, or 21. In some embodiments, VEGF may be added to the medium in the range of 0.1 to 500 ng / ml, preferably 10 to 100 ng / ml, and even more preferably 20 to 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 medium from day 3 to day 14, or for longer periods, such as day 15, 16, 17, 18, 19, 20, or 21. In some embodiments, an HSC cocktail may be added to the medium from day 6 to day 21. The HSC cocktail may contain one or more of SCF, IL-6, IL-3, FLT3L, IGF-1, IL-11, and EPO.The ranges of components of the HSC cocktail are described above. In one embodiment, the HSC cocktail contains 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO. In some embodiments, the EHT cocktail may be added to the culture medium after hypoxic conditions in StemPro-34 medium (e.g., from day 9) until day 14 and beyond, and the EHT cocktail may be changed daily. The EHT cocktail may contain one or more of BMP4 in a concentration range of 1 to 200 ng / ml, SHH in a concentration range of 1 to 200 ng / ml, angiotensin II in a concentration range of 0.1 to 100 μg / ml, and / or losartan potassium in a concentration range of 1 μM to 1000 μM. In some embodiments, SAG may be added in place of or in addition to SHH at a concentration ranging from 1 to 200 ng / ml, preferably 10 ng / ml. In one embodiment, the EHT cocktail contains 10 ng / ml BMP4, 10 ng / ml SHH, 10 μg / ml angiotensin II, and 100 μM losartan potassium, and is changed daily.

[0039] The next step is culturing the cells in StemPro-34 medium under non-hypoxic expansion conditions, where the O concentration is greater than 10% and up to 30%, preferably at normoxic levels or 15-20%, and the CO concentration is between 1% and 10%, preferably 5%, at 32-39°C, preferably 37°C. In some embodiments, the cells are cultured in StemPro-34 medium under non-hypoxic expansion conditions with the addition of HSC cocktail alone. The range of components of the HSC cocktail is described above. In some embodiments, the cells are cultured in StemPro-34 medium under non-hypoxic expansion conditions without the addition of EHT cocktail, VEGF, or bFGF. In some embodiments, the HSC cocktail is replaced every three 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, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO. After this step, HSCs are produced. In some embodiments, the HSCs express CXCR4 on their cell surface.

[0040] For example, the method of the present invention may comprise the steps of: (a) obtaining a population of pluripotent stem cells; (b) culturing on day 0 in SFD medium, 10 μM Y-27632, 10 ng / ml BMP4, and 25 ng / ml bFGF; culturing for 1-2 days in SFD medium, 10 ng / ml BMP4, 5 ng / ml bFGF, and 8 μM CHIR99021; culturing for 1 day in StemPro-34 medium, 12.5 ng / ml bFGF, and 25 ng / ml VEGF; culturing for 1-2 days in StemPro-34 medium, 12.5 ng / ml bFGF, and 25 ng / ml VEGF; culturing for 2-4 days in StemPro-34 medium, 12.5 ng / ml bFGF, 25 ng / ml VEGF, 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml For 3 to 5 days, they were cultured in StemPro-34 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, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, 2U / ml EPO, 10 ng / ml BMP4, 10 ng / ml SHH, 10 μg / ml angiotensin II, and 100 μM losartan potassium, changing the medium daily; for 5 to 10 days, they were cultured in StemPro-34 medium, 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml FLT3L, 25 ng / ml Inducing hematopoietic differentiation by culturing the cells in IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO, changing the medium every three days.

[0041] In one embodiment, the method induces hematopoietic differentiation and generates lin-CD34+CD43+CD45+ cells. In some embodiments, hematopoietic cells and precursors of hematopoietic cells produced as part of the present invention include cells that express 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 that express CD90, cells that express Runx1c, or any combination of the above. Runx1 is an essential gene for hematopoietic development, since deletion of Runx1 causes embryonic lethality. It has also been suggested that the Runx1c isoform is more specifically expressed during definitive hematopoiesis, whereas Runx1a / b is more widely expressed (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).

[0042] The present invention as defined above can also be used to generate cells of myeloid lineage from iPSCs. For example, iPSCs can be obtained as described in Yu et al. (2007) Science 318:1917-1920, and then differentiated into cells enriched for lin-CD34+CD43+CD45+ myeloid progenitor cells. Starting from this point, the above protocol can then be used.

[0043] In some embodiments, the present invention provides for the generation of definitive hematopoietic and long-term repopulating HSCs. In some embodiments, these long-term repopulating HSCs include cells that express 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 that express CD90, cells that express Runx1c, or any combination of the above. Without being bound by theory, expression of CXCR4 is involved in the homing of HSCs and long-term populations of HSCs to the bone marrow. In some embodiments, the HSCs of the present invention include HSCs generated using the methods of the present invention, which express CXCR4 on their cell surface.

[0044] The present invention has been described above with respect to its preferred embodiments. Other forms of the concept are also intended to be within the scope of the following claims.

[0045] Uses of hematopoietic cells and their precursors The hematopoietic cells and hematopoietic progenitor cells provided by the methods and compositions of certain embodiments of the present invention can be used in a variety of applications, including, but not limited to, in vivo transplantation or implantation of hematopoietic cells and hematopoietic progenitor cells, in vitro screening of cytotoxic compounds, carcinogens, mutagens, growth / regulatory factors, pharmaceutical compounds, etc., elucidating the mechanisms of blood disease and injury, studying the mechanisms by which drugs and / or growth factors act, diagnosing and monitoring cancer in patients, gene therapy, and manufacturing biologically active products, to name a few.

[0046] Hematopoietic cells and hematopoietic progenitor cells induced by the programming of the present invention can be used to screen factors (such as solvents, small molecule drugs, peptides, and polynucleotides) or environmental conditions (such as culture conditions or manipulations) that affect the properties of the hematopoietic cells provided herein.

[0047] In some applications, stem cells (differentiated or undifferentiated) are used to screen for factors that promote maturation of cells along the hematopoietic lineage or factors that promote the proliferation and maintenance of such cells in long-term culture. For example, candidate hematopoietic cell maturation or growth factors are tested by adding them to stem cells in different wells, and then any resulting phenotypic changes are assessed according to criteria desired for further culture and use of the cells.

[0048] A particular screening application of the present invention relates to the testing of pharmaceutical compounds in drug research. The reader is generally referred to the standard textbook, *In Vitro Methods in Pharmaceutical Research*, Academic Press, 1997, and U.S. Pat. No. 5,030,015. In certain embodiments of the present invention, cells programmed to the hematopoietic lineage serve as test cells for standard drug screening and toxicity assays, such as those previously performed on hematopoietic cells and hematopoietic progenitor cells in short-term culture. Evaluating the activity of a candidate pharmaceutical compound generally involves combining hematopoietic cells or hematopoietic progenitor cells provided in certain embodiments of the present invention with a candidate compound, determining any changes in cell morphology, marker phenotype, or metabolic activity (compared to untreated cells or cells treated with an inactive compound) that can be attributed to the compound, and then correlating the effect of the compound with the observed changes. Screening may be performed either because the compound is designed to have a pharmacological effect on hematopoietic cells or hematopoietic progenitor cells, or because a compound designed to have an effect elsewhere may have an unintended effect on hematopoietic cells or hematopoietic progenitor cells. Two or more drugs can be tested in combination (by combining with the cells simultaneously or sequentially) to detect possible drug-drug interaction effects.

[0049] The present invention also provides the use of hematopoietic cells and hematopoietic progenitor cells provided herein to restore a degree of function to a subject in need of treatment, possibly due to a blood disease or disorder or injury. For example, hematopoietic cells and hematopoietic progenitor cells derived by the methods disclosed herein can be used to treat blood diseases and disorders, such as hemoglobinopathies and anemias. Furthermore, hematopoietic cells and their precursors can be useful for providing blood or blood cells (e.g., red blood cells, platelets, and neutrophil granulocytes) to subjects in need thereof (e.g., subjects requiring blood transfusions or subjects with blood disorders). Such cells can be useful for treating hematopoietic cell deficiencies resulting from cytosuppressive therapies, such as chemotherapy.

[0050] To determine the suitability of the hematopoietic cells and hematopoietic progenitor cells provided herein for therapeutic applications, the cells can first be tested in a suitable animal model. At one level, the cells are evaluated for their ability to survive and maintain their phenotype in vivo. The programmed cells provided herein are administered to an immunocompromised animal (such as an NOG mouse or an animal rendered immunocompromised chemically or by irradiation) at a suitable site for further observation, such as under the kidney capsule, in the spleen, in the liver lobule, or in the bone marrow. After several days to weeks or longer, tissues are harvested and evaluated for the presence or absence of starting cell types, such as pluripotent stem cells. This can be accomplished by tagging the administered cells with a detectable label (such as green fluorescent protein or β-galactosidase) or by measuring a constitutive marker specific to the administered human cells. When the programmed cells provided herein are tested in rodent models, the presence and phenotype of the administered cells can be assessed by immunohistochemistry or ELISA using human-specific antibodies, or by RT-PCR analysis using primers and hybridization conditions that allow amplification specific for human polynucleotide sequences. Suitable markers for assessing gene expression at the mRNA or protein level are described elsewhere in this disclosure.

[0051] In some embodiments, the present invention is described as follows.

[0052] Embodiment 1. The following steps: a) obtaining a population of pluripotent stem cells; b) culturing the cells under a first hypoxic condition in a supplemented serum-free differentiation (SFD) medium on day 0; c) culturing the cells under a second hypoxic condition in StemPro-34 medium; d) culturing the cells under non-hypoxic conditions in StemPro-34 medium; and e) culturing the cells under non-hypoxic expansion conditions in StemPro-34 medium; and f) Recovering the population of hematopoietic progenitor cells A method for producing hematopoietic progenitor cells, comprising:

[0053] Embodiment 2. The method of embodiment 1, wherein the pluripotent stem cells are human pluripotent stem cells.

[0054] Embodiment 3. The method of embodiment 2, wherein the pluripotent stem cells are induced pluripotent stem cells.

[0055] Embodiment 4 The method of embodiment 2, wherein the pluripotent stem cells are embryonic stem cells.

[0056] Embodiment 5. The method of embodiment 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.

[0057] Embodiment 6. The method of embodiment 5, wherein the BMP4 is in the range of 0.1 to 500 ng / ml.

[0058] Embodiment 7. The method of embodiment 5, wherein BMP4 is added to the culture medium on day 0, day 1, or day 2.

[0059] Embodiment 8 The method of embodiment 5, wherein BMP4 is added to the culture medium on days 0, 1, and 2.

[0060] Embodiment 9. The method of embodiment 5, wherein the bFGF is in the range of 1 to 500 ng / ml.

[0061] Embodiment 10 The method of embodiment 5, wherein bFGF is added to the culture medium on day 0, day 1, or day 2.

[0062] Embodiment 11 The method of embodiment 5, wherein bFGF is added to the culture medium on days 0, 1, and 2.

[0063] Embodiment 12. The method of embodiment 5, wherein Y-27632 is in the range of 100 nM to 30 μM.

[0064] Embodiment 13. The method of embodiment 5, wherein Y-27632 is added to the medium on day 0.

[0065] Embodiment 14. The method of embodiment 5, wherein CHIR99021 is in the range of 0.1 to 20 μM.

[0066] Embodiment 15. The method of embodiment 5, wherein CHIR99021 is added to the medium on day 0, day 1, or day 2.

[0067] Embodiment 16 The method of embodiment 5, wherein CHIR99021 is added to the medium on days 1 and 2.

[0068] Embodiment 17. The method of embodiment 5, wherein SB-431542 is in the range of 0.1 to 20 μM.

[0069] Embodiment 18. The method of embodiment 5, wherein SB-431542 is added to the medium on day 0, day 1, or day 2.

[0070] Embodiment 19. The method of embodiment 5, wherein SB-431542 is added to the medium on day 2.

[0071] Embodiment 20. The method of embodiment 5, wherein BMP4, bFGF, Y-27632, CHIR99021, and SB-431542 are added to the culture medium.

[0072] Embodiment 21. The method of embodiment 20, wherein BMP4 is in a concentration range of 5 to 25 ng / ml and is added to the culture medium on days 0, 1, and 2, bFGF is in a concentration range of 20 to 50 ng / ml and is added to the culture medium on days 0, 1, and 2, Y-27632 is in a concentration range of 5 μM to 20 μM and is added to the culture medium on day 0, CHIR99021 is in a concentration range of 5 μM to 20 μM and is added to the culture medium on days 1 and 2, and SB-431542 is in a concentration range of 0.1 to 20 μM and is added to the culture medium on day 2.

[0073] Embodiment 22. The method of embodiment 21, wherein BMP4 is at a concentration of 10 ng / ml and is added to the culture medium on days 0, 1, and 2, bFGF is at a concentration of 25 ng / ml and is added to the culture medium on days 0, 1, and 2, Y-27632 is at a concentration of 10 μM and is added to the culture medium on day 0, CHIR99021 is at a concentration range of 5 μM to 20 μM and is added to the culture medium on days 1 and 2, and SB-431542 is at a concentration range of 0.1 to 20 μM and is added to the culture medium on day 2.

[0074] Embodiment 23. The method of embodiment 1, wherein the StemPro-34 medium under the second hypoxic condition is supplemented with one or more of bFGF, HSC cocktail, SB-431542, and VEGF, which are added to the StemPro-34 medium under the second hypoxic condition.

[0075] Embodiment 24. The method of embodiment 23, wherein the bFGF is in the range of 20 to 50 ng / ml.

[0076] Embodiment 25 The method of embodiment 23, wherein bFGF is added to the culture medium from day 3 to day 14.

[0077] Embodiment 26. The method of embodiment 23, wherein the HSC cocktail comprises one or more of SCF, IL-6, IL-3, FLT3L, IGF-1, IL-11, and EPO.

[0078] Embodiment 27. The method of embodiment 23, wherein the HSC cocktail comprises SCF, IL-6, IL-3, FLT3L, IGF-1, and / or IL-11, each at a concentration range of 1 to 200 ng / ml, and / or EPO at a concentration range of 0.1 to 20 U / ml.

[0079] Embodiment 28. The method of embodiment 23, wherein the HSC cocktail comprises 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO.

[0080] Embodiment 29. The method of embodiment 23, wherein the HSC cocktail is added to the culture medium from day 6 to day 21.

[0081] Embodiment 30. The method of embodiment 23, wherein SB-431542 is in the range of 0.1 to 20 μM.

[0082] Embodiment 31. The method of embodiment 23, wherein SB-431542 is added to the culture medium from day 3 to day 9.

[0083] Embodiment 32. The method of embodiment 23, wherein the VEGF is in the range of 20 to 50 ng / ml.

[0084] Embodiment 33 The method of embodiment 23, wherein VEGF is added to the culture medium from day 3 to day 14.

[0085] Embodiment 34 The method of embodiment 23, wherein bFGF, HSC cocktail, SB-431542, and VEGF are added to the culture medium.

[0086] Embodiment 35. The method of embodiment 34, wherein bFGF is at a concentration range of 20-50 ng / ml and is added to the culture medium from day 3 to day 14; the HSC cocktail comprises 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO and is added to the culture medium from day 6 to day 21; SB-431542 is at a concentration range of 0.1-20 μM and is added to the culture medium from day 3 to day 9; and VEGF is at a concentration range of 20-50 ng / ml and is added to the culture medium from day 3 to day 14.

[0087] Embodiment 36. The method of embodiment 34, wherein bFGF is at a concentration of 12.5 ng / ml and is added to the culture medium on days 3 to 9, the HSC cocktail comprises 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO and is added to the culture medium on days 6 to 9, SB-431542 is at a concentration of 6 μM and is added to the culture medium on day 3, and VEGF is at a concentration of 25 ng / ml and is added to the culture medium on days 3 to 9.

[0088] Embodiment 37. The method of embodiment 1, wherein the StemPro-34 medium under non-hypoxic conditions is supplemented with one or more of bFGF, HSC cocktail, VEGF, and EHT cocktail, which are added to the StemPro-34 medium under non-hypoxic conditions.

[0089] Embodiment 38. The method of embodiment 37, wherein the bFGF is in the range of 10 to 25 ng / ml.

[0090] Embodiment 39. The method of embodiment 37, wherein bFGF is added to the culture medium from day 3 to day 14.

[0091] Embodiment 40. The method of embodiment 37, wherein bFGF is added to the culture medium from day 9 to day 14.

[0092] Embodiment 41. The method of embodiment 37, wherein the HSC cocktail comprises at least one of SCF, IL-6, IL-3, FLT3L, IGF-1, IL-11, and EPO.

[0093] Embodiment 42. The method of embodiment 37, wherein the HSC cocktail comprises SCF, IL-6, IL-3, FLT3L, IGF-1, and / or IL-11, each at a concentration range of 1 to 200 ng / ml, and / or EPO at a concentration range of 0.1 to 20 U / ml.

[0094] Embodiment 43. The method of embodiment 37, wherein the HSC cocktail comprises 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO.

[0095] Embodiment 44. The method of embodiment 37, wherein the HSC cocktail is added to the culture medium from day 6 to day 21.

[0096] Embodiment 45. The method of embodiment 37, wherein the VEGF is in the range of 20 to 50 ng / ml.

[0097] Embodiment 46 The method of embodiment 37, wherein VEGF is added to the culture medium from day 3 to day 14.

[0098] Embodiment 47. The method of embodiment 37, wherein the EHT cocktail comprises at least one of BMP4, SHH, angiotensin II, and losartan potassium.

[0099] Embodiment 48. The method of embodiment 37, wherein the EHT cocktail comprises BMP4 in a concentration range of 1 to 200 ng / ml, SHH in a concentration range of 1 to 200 ng / ml, angiotensin II in a concentration range of 0.1 to 100 μg / ml, and / or losartan potassium in a concentration range of 1 μM to 1000 μM.

[0100] Embodiment 49. The method of embodiment 37, wherein the EHT cocktail is added to the culture medium from day 9 to day 14.

[0101] Embodiment 50 The method of embodiment 23, wherein bFGF, HSC cocktail, VEGF, and EHT cocktail are added to the culture medium.

[0102] Embodiment 51. The method of embodiment 34, wherein bFGF is in a concentration range of 10-25 ng / ml and is added to the culture medium from day 3 to day 14, the HSC cocktail comprises 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, and is added to the culture medium from day 6 to day 21, VEGF is in a concentration range of 20-50 ng / ml and is added to the culture medium from day 3 to day 14, and the EHT cocktail comprises BMP4, SHH, angiotensin II, and losartan potassium and is added to the culture medium from day 9 to day 14.

[0103] Embodiment 52. The method of embodiment 34, wherein bFGF is at a concentration of 12.5 ng / ml and is added to the culture medium on days 9 to 14, the HSC cocktail comprises 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO and is added to the culture medium from days 6 to 21, VEGF is at a concentration of 12.5 ng / ml and is added to the culture medium on days 9 to 14, and the EHT cocktail comprises BMP4, SHH, angiotensin II, and losartan potassium and is added to the culture medium on days 9 to 14.

[0104] Embodiment 53 The method of embodiment 1, wherein the StemPro-34 medium under non-hypoxic expansion conditions is supplemented with an HSC cocktail that is added to the StemPro-34 medium under non-hypoxic expansion conditions.

[0105] Embodiment 54. The method of embodiment 53, wherein the HSC cocktail comprises at least one of SCF, IL-6, IL-3, FLT3L, IGF-1, IL-11, and EPO.

[0106] Embodiment 55. The method of embodiment 53, wherein the HSC cocktail comprises SCF, IL-6, IL-3, FLT3L, IGF-1, and / or IL-11, each in a concentration range of 1 to 200 ng / ml, and / or EPO in a concentration range of 0.1 to 20 U / ml.

[0107] Embodiment 56. The method of embodiment 53, wherein the HSC cocktail comprises 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO.

[0108] Embodiment 57. The method of embodiment 53, wherein the HSC cocktail is added to the culture medium from day 6 to day 21.

[0109] Embodiment 58 The method of embodiment 1, wherein the first hypoxic condition contains an O2 concentration of less than 10%.

[0110] Embodiment 59. The method of embodiment 1, wherein the second hypoxic condition contains an O2 concentration of less than 10%.

[0111] Embodiment 60. The method of embodiment 1, wherein culturing the cells under non-hypoxic conditions in StemPro-34 medium contains an O2 concentration greater than 10%.

[0112] Embodiment 61 The method of embodiment 1, wherein culturing the cells under non-hypoxic expansion conditions in StemPro-34 medium contains an O2 concentration greater than 10%.

[0113] Embodiment 62. A method for producing hematopoietic progenitor cells from pluripotent stem cells or from transdifferentiation of somatic cells, comprising culturing pluripotent stem cells or somatic cells under conditions to generate hematopoietic progenitor cells capable of differentiating into various hematopoietic lineage cells, the method comprising the steps of: (a) obtaining a population of pluripotent stem cells; (b) 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 in SFD medium, 10 ng / ml BMP4, 5 ng / ml bFGF, and 8 uM CHIR99021; culturing for 1 day in StemPro-34 medium, 12.5 ng / ml bFGF, and 25 ng / ml VEGF; culturing for 1-2 days in StemPro-34 medium, 12.5 ng / ml bFGF, and 25 ng / ml VEGF. Culture in VEGF; StemPro-34 medium, 12.5ng / ml bFGF, 25ng / ml VEGF, 50ng / ml SCF, 25ng / ml IL-6, 25ng / ml IL-3, 25ng / ml FLT3L, 25ng / ml IGF-1, 5ng / ml IL-11, and 2U / ml for 2-4 days. Cultured in EPO; StemPro-34 medium, 12.5ng / ml bFGF, 12.5ng / ml VEGF, 50ng / ml SCF, 25ng / ml IL-6, 25ng / ml IL-3, 25ng / ml FLT3L, 25ng / ml IGF-1, 5ng / ml IL-11, 2U / ml EPO, 10ng / ml BMP4, 10ng / ml for 3-5 days. culturing the cells in SHH, 10 μg / ml angiotensin II, and 100 μM losartan potassium, changing the medium every day; and inducing hematopoietic differentiation by culturing the cells in StemPro-34 medium, 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO, changing the medium every three days for 5 to 10 days.

[0114] Embodiment 63. The method of embodiment 62, wherein the medium having StemPro-34 medium, 12.5 ng / ml bFGF, and 25 ng / ml VEGF further comprises 6 uM SB431542.

[0115] Embodiment 64. The method of embodiment 62 or 63, wherein the medium further comprises 6 μM SB431542 (TOCRIS) on day 2, day 3, day 4, or day 5.

[0116] Embodiment 65. The method of any one of embodiments 1 to 64, wherein the pluripotent stem cells are induced pluripotent stem cells.

[0117] Embodiment 66 The method of any one of embodiments 1 to 64, wherein the pluripotent stem cells are embryonic stem cells.

[0118] Embodiment 67 The method of any one of embodiments 1 to 66, wherein the pluripotent stem cells are capable of homing to the bone marrow.

[0119] Embodiment 68. The method of embodiment 67, wherein the hematopoietic progenitor cells express CXCR4.

[0120] Embodiment 69. The method of embodiment 68, wherein the hematopoietic progenitor cells express CXCR4 on the cell surface.

[0121] Embodiment 70. The method of any one of embodiments 1 to 69, wherein the hematopoietic progenitor cells are CD34+, CD45+, CD90+, or THY1+.

[0122] Embodiment 71. The method of embodiment 70, wherein the hematopoietic progenitor cells are CD34+, CD45+, CD90+ and THY1+.

[0123] Embodiment 72. The method of any one of embodiments 1 to 71, wherein the hematopoietic progenitor cells are CD38-, Lin-, CD43-, or CD73-.

[0124] Embodiment 73. The method of embodiment 72, wherein the hematopoietic progenitor cells are CD38-, Lin-, CD43-, and CD73-.

[0125] Embodiment 74. The method of any one of embodiments 1 to 73, wherein the hematopoietic progenitor cells are CD45+, CD34+, CD90+, CD38-, and Lin-.

[0126] Embodiment 75. The method of any one of embodiments 1 to 74, wherein the hematopoietic progenitor cells are CD90+.

[0127] Embodiment 76 The method of any one of embodiments 1 to 75, wherein the hematopoietic progenitor cells express Runx1c.

[0128] Embodiment 77. Hematopoietic progenitor cells produced using any of the methods described in embodiments 1 to 76.

[0129] Embodiment 78. Hematopoietic progenitor cells according to embodiment 77, capable of long-term engraftment in bone marrow.

[0130] Incorporation by Reference All publications, patents, and patent applications mentioned herein are 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 admission that such reference is prior art to the present invention. In the event that any definitions or terms provided in a reference incorporated by reference differ from the terms and discussion provided herein, the terms and definitions in this specification shall control.

[0131] equivalent The foregoing specification is believed 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 set forth the best mode contemplated by the inventors. However, it will be appreciated that no matter how detailed the foregoing appears in text, the invention can be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0132] The following examples, including the experiments conducted and results obtained, are provided for illustrative purposes only and are not to be construed as limiting the invention. [Example]

[0133] Example 1: Process of hematopoietic stem cell generation iPSCs were added to 6-well plates coated with 1 ml of a 1:7 dilution of poly-L-ornithine (PLO; Sigma) in PBS and incubated for 2 hours at 37°C. The PLO solution was replaced with 1 ml of a 1:150 dilution of laminin (Sigma) in DMEM / F12 and incubated for 2 hours at 37°C.

[0134] On day 0, iPSCs were lifted using TrypLE (Thermo Fisher) and 600,000 cells per well were seeded in 2 ml of SFD medium (75:25 IMDM:Ham's F-12, 0.05% BSA, 1x B27, 0.5x N2 supplement, 1x GlutaMax and 1x penicillin-streptomycin, 0.5 mM ascorbic acid, 450 μM monothioglycerol, and 150 μg / mL holo-transferrin (R&D Systems)) + 10 μM Y-27632 + 10 ng / ml BMP4 + 25 ng / ml bFGF.

[0135] On day 1, the medium was changed to SFD medium + 10 ng / ml BMP4 + 25 ng / ml bFGF + 8 μM CHIR99021, and 2 ml was added to each well. On days 2, 3, 4, and 5, 6 μM SB-431542 (TOCRIS) was added to the medium in some samples. On day 3, the medium was changed to StemPro-34 medium + 12.5 ng / ml bFGF + 25 ng / ml VEGF + 6 μM SB431542, and 2 ml was added to each well and incubated for 24 hours. On day 4, the medium was changed to StemPro-34 medium + 12.5 ng / ml bFGF + 25 ng / ml VEGF, and 2 ml was added to each well and incubated for 48 hours. On days 6–8, the medium was replaced with StemPro-34 medium + 12.5 ng / ml bFGF + 25 ng / ml VEGF + 50 ng / ml SCF + 25 ng / ml IL-6 + 25 ng / ml IL-3 + 25 ng / ml FLT3L + 25 ng / ml IGF-1 + 5 ng / ml IL-11 + 2 U / ml EPO, and 2 ml was added to each well. On days 9-13, the medium was changed to StemPro-34 medium + 12.5ng / ml bFGF + 12.5ng / ml VEGF + 50ng / ml SCF + 25ng / ml IL-6 + 25ng / ml IL-3 + 25ng / ml FLT3L + 25ng / ml IGF-1 + 5ng / ml IL-11 + 2U / ml EPO + 10ng / ml BMP4 + 10ng / ml SHH + 10ug / ml angiotensin II + 100uM losartan potassium, 2ml was added to each well, and the medium was changed every 2-3 days. From days 14 to 21, the medium was changed to StemPro-34 medium + 50 ng / ml SCF + 25 ng / ml IL-6 + 25 ng / ml IL-3 + 25 ng / ml FLT3L + 25 ng / ml IGF-1 + 5 ng / ml IL-11 + 2 U / ml EPO, 2 ml of which was added to each well. The medium was changed every 3 days. On day 21, cells were FACS-sorted to obtain Lin- (CD45RA, CD10, CD7, CD3, CD19, CD33, CD66b) CD34+ CD45+ CD38- CD90+ cells. From days 0 to 10, cells were incubated at 37°C, 5% O2, and 5% CO2. From days 11 to 21, cells were incubated at 37°C, 20% O2, and 5% CO2.

[0136] Example 2: Assay for the presence of hemogenic endothelial cells and hematopoietic stem cells Using the protocol described in Example 1, cells on days 9 and 10 of culture were sequenced using single-cell sequencing. Hemogenic endothelial cells are a subset of endothelial cells that can differentiate into hematopoietic cells. Hemogenic endothelial cells are characterized as CD34+THY1+CD43-CD73-. FACS plots demonstrating the presence of hemogenic endothelial cells are shown in Figure 1 for both the previous protocol and the current protocol described in Example 1. Further analysis of hematopoietic stem cells (CD34+CD45+CD73-) indicated the time window for the endothelial-to-hematopoietic transition in iPSC cultures as days 19-21 of differentiation. These results are shown in Figure 2.

[0137] Example 3: Limiting dilution assay to measure the pluripotency of HSCs derived from iPSCs Putative HSCs (CD34+CD45+CD90+CD38-Lin-) derived from iPSCs were purified using FACS. Cells were plated at 20, 10, 5, 2, or 1 cell / well, and each well was plated with methylcellulose along with permissive cytokines. Cells were cultured for 14 days, and colonies were scored for colony-forming units. The results are shown in Figure 3. Figure 3A shows the percentage of wells containing each cell type when different numbers of cells were plated. Depending on the number of cells plated per well, different cell fractions formed colonies, including erythroid burst-forming units (BFU-E), macrophage colony-forming units (CFU-M), granulocyte-macrophage CFUs (CFU-GM), eosinophil colony-forming units (CFU-E), granulocyte CFUs (CFU-G), and pluripotent CFUs (CFU-GEMM), as shown in Figure 3B.

[0138] Example 4: Generation of Runx1C-GFP gene reporter system Runx1 is an essential gene for hematopoiesis, because its deletion causes embryonic lethality. It has also been suggested that the Runx1c isoform is more specifically expressed during definitive hematopoiesis, whereas Runx1a / b is more widely expressed (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).

[0139] The purpose of creating the GFP-2A-Runx1c gene reporter line was to fluorescently label newborn hematopoietic stem cells (HSCs) emerging from hemogenic endothelial cells, enabling analysis of Runx1c expression, which provided a direct readout of the efficiency of our HSC differentiation protocol.

[0140] The target design and vector were constructed using the following steps. The N-terminal targeting guide RNA 5'-GCATTTTCAGGAGGAAGCGA-3' (SEQ ID NO: 1) of Runx1c was cloned into the pCas9-Guide vector (ORIGENE) using BamHI / BsmBI. The generation of the GFP-2A-Runx1c hiPSC reporter line for labeling hematopoietic stem cells (HSCs) is shown in Figure 4. The "GFP-2A" sequence was inserted before the ATG start codon of Runx1c exon 1, and a "LoxP-PGK-BSD-pA-LoxP" cassette was also inserted into intron 1 for enrichment of correctly targeted human induced pluripotent stem cell (hiPSC) clones. The homology arms flanking the knock-in sequence consisted of 1 kb upstream and downstream of the guide RNA target site. 7.5ug of pCas9-Ruxn1c-Guide vector and 7.5ug of GFP-2A-Runx1c donor vector were mixed in 2 x 106 iPSCs were transfected using Lipofectamine 3000. 48 hours after transfection, 2.5 μg / ml blasticidin was applied to enrich the target population. Cells were selected for 5-7 days and expanded for cryopreservation. Figure 4A shows a schematic diagram illustrating the strategy for targeting the Runx1c genomic locus. Meanwhile, 1 x 10 cells enriched by blasticidin were 6 iPSCs were harvested for genomic DNA isolation and PCR genotyping. Figure 4B shows that the primers described in Figure 4A were used to screen positive colonies after genome editing. After blasticidin selection, a total of 48 single-cell clones were harvested, expanded, and subjected to PCR genotyping analysis. Thirty-eight clones showed positive genotyping bands on the agarose gel (efficiency = 79%). Figure 4C shows an image of selected positive clones of the GFP-2A-Runx1c hiPSC line.

[0141] The primers in Table 1 below were used for genotyping and sequencing of different regions of the targeted Runx1c locus.

[0142] [Table 1]

[0143] PCR was performed using 100 ng of genomic DNA from the concentrated transfection pool with PfuUltra II Hotstart PCR Master Mix (Agilent). The sequence of the purified PCR product was confirmed by Sanger sequencing (Genewiz).

[0144] For single-cell cloning, blasticidin-resistant iPSCs were dissociated into single cells using TryPLE and seeded at a single-cell density (approximately 2500 cells per 10 cm dish) in mTeSR medium. CloneR (STEMCELL Technologies) was added for the first 4 days to promote the survival and growth of single-cell clones. A second round of blasticidin selection was applied on days 4–7 to further enrich for specifically targeted clones. Around days 8–10, colonies that emerged from single cells were picked under a microscope in a tissue culture cabinet and transferred to Matrigel-coated 96-well plates for continued culture.

[0145] When colonies grew to near confluence in 96-well plates, the cells were detached using ReLeSR (STEMCELL Technologies) and resuspended in mTeSR supplemented with 10 μM Y-27632 (TOCRIS) to passage the plate. The cell suspension was then split into 3 x 96-well replicate plates at ratios of 1:3, 1:5, and 1:8, respectively. After several days, the 1:5 plate was split again for cryopreservation.

[0146] When the cells in the 1:3 plates reached full confluence, they were lysed using 50 μl / well of QuickExtract™ DNA Extraction Solution (Lucigen) according to the manufacturer's instructions for PCR screening. For PCR screening, 3 μl of the DNA extraction solution was used as a PCR template with the primer set LH-In-F / GFP-R. Selected PCR-positive colonies were confirmed by PCR and Sanger sequencing using the additional primer sets listed in step 3.

[0147] Confirmed GFP-2A-Runx1c hiPSC clones were expanded from 1:8 replicate plates for downstream applications. Our data showed that the temporal expression of Runx1c-GFP highly overlaps with the existing HSC markers CD34 and CD45, but marks only a subpopulation of the CD34 / CD45 double-positive population (see Figure 6). Thus, Runx1c-GFP serves as an additional marker for further purifying HSC populations to enhance purity and efficacy.

[0148] Figure 5 shows visualization of GFP-positive hematopoietic stem cells during differentiation of hiPSCs. GFP-2A-Runx1c iPSCs (day 0, upper left panel) first differentiated into endothelial cells (day 9, upper right panel), and then underwent endothelial-to-hematopoietic transition (EHT). GFP-positive HSCs emerged from selected regions of the GFP-negative endothelial layer (dashed frame, "blood islands") (day 14, middle panel). By day 17, the production of GFP-positive HSCs was no longer restricted to specific regions but became more prominent throughout the tissue culture (day 17, bottom panel).

[0149] Figure 6 shows the time course of surface marker expression patterns of GFP-2A-Runx1c iPSCs during hematopoietic differentiation. (A) Single-positive population. (B) Runx1c+CD34+CD45+ putative hematopoietic stem cell population.

[0150] Example 5: Long-term iPSC cell marker expression assay Time course of CD34 and GFP-Runx1c expression LT-iPSCs and LT-iPSCs stably expressing GFP-Runx1c were differentiated using the protocol described in Example 1. Adherent cells on day 9 and suspension cells on days 14, 16, 17, 20, and 21 were collected for FACS analysis. All sample groups for FACS were stained with APC-CD34 and SYTOX Blue (Thermo Fisher). FACS analysis was performed by gating on single cells using negative SYTOX Blue staining. Figure 7 shows the expression of CD34 versus GFP-Runx1c in HSCs on days 9 and 14. Figure 8 shows the expression of CD34 versus GFP-Runx1c in HSCs on days 16 and 17. Figure 9 shows the expression of CD34 versus GFP-Runx1c in HSCs on days 20 and 21. From left to right, each figure shows LT-iPSCs, iPSCs overexpressing GFP-Runx1c, and a superposition of both. GFP-Runx1c expression began on day 14 and increased over time. From days 14 to 17, all GFP-Runx1c-positive cells were CD34+. From day 20, GFP-Runx1c cells became CD34-.

[0151] Expression of different cell populations over time Various HSC populations were purified by flow cytometry (FACS) sorting. Five thousand HSCs from each population were cultured in 5 ml MethoCult™ H4435 Enriched (STEMCELL Technologies Inc.) in 6-well plates at 37°C and 5% CO2. After 21 days of culture, all cells in MethoCult™ were collected and diluted in DMEM / F12. After centrifugation at 1000 g for 5 minutes, the cell pellet was titrated repeatedly using a P1000 pipette, and the number of single cells was counted using a ViaCell™. HSCs derived from LT-iPSCs and GFP-Runx1c iPSCs were sorted based on the gating strategy described above. On days 16, 17, and 20, only LT:CD45+ / CD34+ cells were sorted from LT-iPSCs and Runx1c:CD34+ / GFP- cells, and Runx1c:CD34+ / GFP- cells were sorted. On day 21, all six populations were sorted for CFU assays, as shown in Figure 10. Figure 10 shows the sorting of cell populations for CFU assays from LT-iPSCs and GFP-Runx1c iPSCs. For all HSCs from Runx1c-GFP, all CD45+ cells were first gated. All populations were CD45+ cells. At early stages, HSCs expressing GFP-Runx1c generated equal or fewer total CFUs, but on day 21, HSCs GFP-Runx1c and CD34+ double-positive HSCs were more robust in generating more cells from CFUs (as shown in Figure 11). In all groups, CD34+ is essential for maintaining CFU potency.

[0152] Cell type marker analysis HSCs cultured for 21 days in MethoCult™ medium as described above were harvested, titrated into single-cell suspensions, blocked with 1% BSA and FcR receptor blockers, stained with antibodies, and subjected to FACS analysis to check for expression of surface markers for all lineages. Figure 12 shows a CFU panel of common progenitor markers. Day 16 HSCs, which begin to show strong Runx1c expression, maintain multiple common progenitor markers after being cultured to CFUs. As HSCs become more mature, CD34+ cells show a decrease in Runx1c expression, and cells from CFUs show minimal common progenitor markers. Figure 13 shows a CFU panel of lymphoid markers. Although MethoCult™ was designed to expand myeloid cells in vitro, a small proportion of lymphoid lineage cells were identified within the CFUs, including T cells, B cells, and NK cells. Day 16 HSCs have been shown to be more potent than day 21 HSCs in generating lymphoid lineage cells. Figure 14 shows a CFU panel of myeloid markers. HSCs at all stages demonstrate robust potential for generating myeloid lineage cells in CFU assays. All myeloid lineage cells, except platelets, were identified among the CFUs of CD34+ HSC cells.

Claims

1. a) obtaining a population of pluripotent stem cells; b) culturing the cells under a first hypoxic condition in supplemented serum-free differentiation (SFD) medium on day 0; c) culturing the cells under a second hypoxic condition in StemPro-34 medium; d) culturing the cells under non-hypoxic conditions in StemPro-34 medium; e) culturing the cells under non-hypoxic expansion conditions in StemPro-34 medium; and f) Recovering the population of hematopoietic progenitor cells A method for producing hematopoietic progenitor cells, comprising:

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

3. 10. 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 to 25 ng / ml and is added to the culture medium on days 0, 1, and 2, the bFGF is at a concentration range of 20 to 50 ng / ml and is added to the culture medium on days 0, 1, and 2, the Y-27632 is at a concentration range of 5 μM to 20 μM and is added to the culture medium on day 0, the CHIR99021 is at a concentration range of 5 μM to 20 μM and is added to the culture medium on days 1 and 2, and the SB-431542 is at a concentration range of 0.1 to 20 μM and is added to the culture medium on day 2.

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

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

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

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

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

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

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

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

13. 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, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO.

14. The first hypoxic condition is less than 10% O ２ The method of claim 1 , comprising:

15. The second hypoxic condition is less than 10% O ２ The method of claim 1 , comprising:

16. 1. A method for producing hematopoietic progenitor cells from pluripotent stem cells or from transdifferentiation of somatic cells, comprising culturing the pluripotent stem cells or somatic cells under conditions to generate hematopoietic progenitor cells capable of differentiating into various hematopoietic lineage cells; (a) obtaining a population of pluripotent stem cells; (b) On day 0, cultured in SFD medium, 10 μM Y-27632, 10 ng / ml BMP4, and 25 ng / ml bFGF; for 1-2 days, cultured in SFD medium, 10 ng / ml BMP4, 5 ng / ml bFGF, and 8 μM CHIR99021; for 1 day, cultured in StemPro-34 medium, 12.5 ng / ml bFGF, and 25 ng / ml VEGF; for 1-2 days, cultured in StemPro-34 medium. For 2-4 days, they were cultured in StemPro-34 medium, 12.5 ng / ml bFGF, 25 ng / ml VEGF, 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO; for 3-5 days, they were cultured in StemPro-34 medium, 12.5 ng / ml bFGF, 25 ng / ml VEGF, 50 ng / ml SCF, 25 ng / ml IL-6, 25 ng / ml IL-3, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, and 2 U / ml EPO; emPro-34 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, 25 ng / ml FLT3L, 25 ng / ml IGF-1, 5 ng / ml IL-11, 2 U / ml EPO, 10 ng / ml BMP4, 10 ng / ml SHH, 10 ug / ml angiotensin II, and 100 uM Losal culturing the cells in a medium containing 50 ng / ml of SCF, 25 ng / ml of IL-6, 25 ng / ml of IL-3, 25 ng / ml of FLT3L, 25 ng / ml of IGF-1, 5 ng / ml of IL-11, and 2 U / ml of EPO for 5 to 10 days, changing the medium every 3 days to induce hematopoietic differentiation.

17. 17. The method of claim 16, wherein the medium having StemPro-34 medium, 12.5 ng / ml bFGF, and 25 ng / ml VEGF further comprises 6 uM SB431542.

18. 17. The method of claim 16, wherein the medium further comprises 6 μm SB431542 (TOCRIS) on day 2, day 3, day 4, or day 5.

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

20. 20. The method of claim 19, wherein the hematopoietic progenitor cells express CXCR4.

21. The method of claim 1, wherein the hematopoietic progenitor cells are CD34+, CD45+, CD90+, or THY1+.

22. The method of claim 1, wherein the hematopoietic progenitor cells are CD38-, Lin-, CD43-, or CD73-.

23. The method of claim 1, wherein the hematopoietic progenitor cells are CD45+, CD34+, CD90+, CD38-, and Lin-.

24. The method of claim 1, wherein the hematopoietic progenitor cells are CD90+.

25. The method of claim 1, wherein the hematopoietic progenitor cells express Runx1c.

26. Hematopoietic progenitor cells produced using any of the methods of claims 1 to 25.

27. The hematopoietic progenitor cells of claim 26, which are capable of long-term engraftment in bone marrow.

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