Myeloid lineage derived from pluripotent cells

Abstract

The present disclosure provides efficient in vitro methods for generating myeloid cell lineages from human induced pluripotent stem cells (iPSCs). The cells produced in various embodiments according to the present disclosure are functional and / or more closely similar to corresponding lineages isolated from peripheral blood, bone marrow, or other tissue. The invention provides, in some aspects, isolated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 413,420, filed on October 5, 2022, the content of which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing that has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. The XML copy, created on September 26, 2023, is named GRU-015PC_Sequence_Listing.xml and is 30,037 bytes in size. BACKGROUND OF THE INVENTION

[0005] Innate myeloid lineage cells play a crucial role in tissue maintenance and contribute to coordinating immune responses. However, their clinical use is hindered by the small numbers of such cells that can be isolated from conventional leukocyte-depleted products. Thus, the development of large-scale, off-the-shelf myeloid lineage cells (such as monocytes, macrophages, dendritic cells, and neutrophils and their precursors) is an attractive immunotherapy to develop as a tool against inflammation, autoimmune diseases, cancer, antimicrobial diseases, and the like. SUMMARY OF THE INVENTION

[0006] The present disclosure provides, in various aspects and embodiments, methods for generating hematopoietic lineages for use in cell therapy, including innate myeloid lineages such as monocytes, macrophages, dendritic cells, and neutrophils, and their precursors. In various embodiments, the present invention provides efficient in vitro methods for developing such hematopoietic lineages from human induced pluripotent stem cells (iPSCs), including gene-edited iPSCs. The cells generated in various embodiments according to the present disclosure are functional and / or more closely resemble the corresponding lineages isolated from peripheral blood or bone marrow. The present invention also provides isolated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

[0007] On the one hand, the present disclosure provides a method for preparing a cell population comprising myeloid cells of the innate immune system. The method includes preparing a population of pluripotent stem cells (PSCs) (such as a population of induced pluripotent stem cells (iPSCs) that differentiate into embryoid bodies), and enriching CD34+ cells to prepare a CD34+-enriched population. Endothelial-to-hematopoietic transition (EHT) is induced in the CD34+-enriched population to prepare a population of hematopoietic stem cells (HSCs), and then optionally further enriching CD34+ cells. The resulting HSC population (or a fraction thereof) can differentiate into myeloid lineages of the innate immune system (e.g., phagocytes or their precursors). In some embodiments, the present disclosure provides a method for generating neutrophils, monocytes / macrophages, and immature and mature myeloid dendritic cells (DCs) (or their precursors) in vitro from a population of HSCs).

[0008] In various embodiments, iPSCs are prepared by reprogramming somatic cells. In some embodiments, iPSCs are generated from somatic cells, such as (but not limited to) fibroblasts or PBMCs (or cells isolated therefrom). In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood.

[0009] In various embodiments, iPSCs are gene-edited to assist in HLA matching, such as deleting one or more HLA class I and / or class II alleles. For example, iPSCs can be gene-edited to delete one or more of HLA-A, HLA-B, and HLA-C, and one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, iPSCs retain the expression of at least one HLA class I and at least one HLA class II complex. In certain embodiments, iPSCs are homozygous for at least one retained class I and class II locus. In some embodiments, iPSCs are gene-edited to be HLA-A neg homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg . In some embodiments, iPSCs are further homozygous for HLA-DRB1.

[0010] In various embodiments, iPSCs are prepared and amplified using a culture system. The amplified iPSCs can be recovered from the culture to generate embryoid bodies (EBs). EBs differentiated from iPSCs are three-dimensional aggregates of iPSCs and contain three (or alternatively two or one) germ layers (depending on the differentiation method). In some embodiments, the method includes harvesting CD34+-enriched cells from the EBs and inducing endothelial-to-hematopoietic differentiation.

[0011] In some embodiments, iPSC differentiation proceeds until the cells are at least about 20% CD34+ or at least about 30% CD34+. In some embodiments, CD34 enrichment and EHT can be induced between day 7 and day 14 of iPSC differentiation. Differentiation of iPSCs can be carried out according to known techniques. In some embodiments, iPSC differentiation involves a combination of factors such as, but not limited to, bFGF, Y27632, BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1.

[0012] Induction of EHT can be carried out by any known method. In some embodiments, induction of EHT produces a population of hematopoietic stem cells (HSCs) containing LT-HSCs. In some embodiments, EHT generates HSCs from endothelial cells or hematopoietic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., by stimulation, inhibition, and / or genetic modification). In some embodiments, EHT produces a stem cell population that contains one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs), and hematopoietic stem cell progenitors.

[0013] In some embodiments, the method includes increasing the expression or activity of DNA (cytosine-5-)-methyltransferase 3β (Dnmt3b) in PSCs, embryoid bodies, CD34+-enriched cells, ECs, HECs, or HSCs, which can be carried out by mechanical, genetic, biochemical, or pharmacological means. For example, in some embodiments, the cells are contacted with an effective amount of a mechanosensor or mechanosensitive channel agonist that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensor is Piezol. An exemplary Piezol agonist is Yoda1. In various embodiments, pharmacological Piezo1 activation is applied to CD34+ cells harvested from EBs. In some embodiments, the method does not involve increasing the expression of dnmt3b, such as by using a Piezo1 agonist.

[0014] In various embodiments, CD34+ cells (e.g., floating cells and / or adherent cells) are harvested from a culture that undergoes an endothelial-to-hematopoietic cell transition between day 10 and day 20 of iPSC differentiation (such as between day 12 and day 17 of iPSC differentiation). Hematopoietic stem cells (HSCs) capable of giving rise to the innate myeloid, erythroid, and lymphoid lineages can be identified based on the expression of CD34 and the absence of lineage-specific markers (referred to as Lin-).

[0015] In various embodiments, the HSC population or its fractions differentiate into hematopoietic lineages, which can be selected from common myeloid progenitors (CMPs), lymphoid-primed multipotent progenitors (LMPPs), granulocyte macrophage DC progenitors (GMDPs), granulocyte / macrophage lineage-restricted progenitors (GMPs), megakaryocyte / erythroid progenitors (MEPs), macrophage / dendritic cell (DC) progenitors (MDPs), common DC progenitors (CDPs), conventional (or classical) myeloid dendritic cells (cDCs), common monocyte progenitors (cMoPs), and plasmacytoid DCs (pDCs) and their fractions, from which monocytes, macrophages, and dendritic cells can be generated.

[0016] In some embodiments, the cells are modified to express a chimeric antigen receptor (CAR). Additionally, or optionally, the phagocytic CAR can be engineered to express a cytokine (e.g., IL-4, IL-6, IL-15, etc. or interferon) to make the CAR-expressing cells more effective in targeting tumors, for example. In non-limiting examples, the cells can be efficiently transduced by a vector, such as but not limited to a retrovirus or non-integrating viral vector or non-viral vector carrying the CAR. In some embodiments, the CAR can target a tumor-associated antigen or marker.

[0017] In other aspects, the present invention provides a cell population or a pharmaceutically acceptable composition thereof comprising myeloid cells or their precursors, and which can be produced by the methods described herein. In some embodiments, the cell population is a progenitor myeloid lineage cell population that is capable of engrafting in the thymus, spleen, or secondary lymphoid organs after administration to a subject in need thereof. In various embodiments, a composition for cell therapy is prepared, which comprises the cell population and a pharmaceutically acceptable excipient. In some embodiments, the cell population is HLA-A neg homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg . In some embodiments, the cell population is further homozygous for HLA-DRB1. In various embodiments, the composition comprises a myeloid lineage selected from one or more of monocytes, macrophages, dendritic cells, neutrophils, and myeloid progenitors.

[0018] In other aspects, the present invention provides a method of cell therapy, comprising administering to a human subject in need thereof the cell population or a pharmaceutically acceptable composition thereof described herein. In various embodiments, the methods described herein are used to treat blood (malignant and non-malignant), bone marrow, immune diseases, and infectious diseases. In various embodiments, the human subject has a condition comprising one or more of lymphopenia, cancer, immunodeficiency, and autoimmune diseases.

[0019] From the following detailed disclosure and working examples, other aspects and embodiments of the present disclosure will become apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Show that overexpression (OE) of ETV2 does not affect pluripotency. Figure 1 Show FACS plots representing the transduction efficiency of overexpressing ETV2 and GFP sequences in iPSCs using an adenoviral vector. As shown by the expression of the TRA-1-60 stem cell marker, overexpression of ETV2 does not affect the stem cell properties of iPSCs.

[0021] Figure 2 Show that overexpression (OE) of ETV2 increases the yield of hematopoietic endothelial cells. Representative flow cytometric analysis and relative quantification of hematopoietic endothelial cells (described as CD235a-CD34+CD31+) indicate that ETV2-OE enhances the formation of hematopoietic endothelial cells.

[0022] Figure 3 Show that overexpression (OE) of ETV2 enhances the formation of CD34+ cells during iPSC differentiation. Representative flow cytometric analysis of CD34+ cells, and relative quantification indicate that ETV2-OE enhances the formation of CD34+ cells.

[0023] Figure 4A and Figure 4B Show that iPSC-derived HSCs activated with Piezo1 undergo pro-T cell differentiation similar to bone marrow (BM)-HSCs. Figure 4A Is a FACS plot of the differentiation efficiency of bone marrow (BM) HSCs and iPSC-HSCs activated with Piezo1 into CD34+CD7+ pro-T cells. Figure 4B Is a quantification of CD34+CD7+ cells (%) derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1 activation). Figure 4B Show the mean of three experiments.

[0024] Figure 5A and Figure 5B Show that iPSC-derived HSCs generated by Piezo1 activation undergo T cell differentiation and can be activated by CD3 / CD28 beads similar to BM-HSCs. Figure 5A Is a FACS plot of the activation efficiency (CD3+CD69+ expression) of T cells differentiated from BM-HSCs and iPSC-derived HSCs generated by Piezo1 activation. Figure 5B Is a quantification of CD3+CD69+ cells (%) derived from (1) BM-HSCs and (2) iPSC-HSCs (Piezo1 activation). Figure 5BDisplays the average value of three experiments.

[0025] Figure 6 Shows that iPSC-derived HSCs (activated with Piezo1 in this example) can differentiate into functional T cells. IFNγ expression is a result of T cell activation after stimulation of the T cell receptor (TCR) via CD3 / CD28 beads. The enhanced expression of IFNγ in T cells differentiated from iPSC-derived HSCs after Piezo1 activation increased the ability of HSCs to further differentiate into functional cells (e.g., T cells, myeloid cells, etc.). Figure 6 Displays the average value of three experiments.

[0026] Figure 7 A and Figure 7 B show that HSCs derived from differentiated iPSCs (D8+7) can differentiate into neutrophils, as identified by the presence of CD15+ and CD11b (neutrophil markers) Figure 7 (A) or the release of myeloperoxidase (MPO) by neutrophils Figure 7 (B), and are superior to D8-iPSC-CD34+ cells in differentiating into neutrophils.

[0027] Figure 8 A and Figure 8 B show that neutrophils differentiated from HSCs derived from differentiated iPSCs (D8+7) have phagocytic activity.

[0028] Figure 9A and Figure 9B Shows the phenotypic analysis of HLA-edited (e.g., triple knockout) cells by FACS and immunofluorescence. Figure 9A Shows the overall expression of HLA class I molecules (HLA-A, HLA-B, and HLA-C) on the cell surface, where HLA-edited cells are positive for overall HLA class I expression to a similar extent as wild-type cells (i.e., gHSCs). Figure 9B Shows the cellular expression of HLA-A via immunofluorescence, where HLA-A is not expressed in HLA-edited clones.

[0029] Figure 10 Shows that HLA-edited clones retain their pluripotency (maintaining tri-lineage differentiation), as shown by immunofluorescence, where ectodermal differentiation is indicated by nestin-488 and PAX6-594 staining, mesodermal differentiation is indicated by GATA-488 staining, and endodermal differentiation is indicated by CXCR4-488 and FOX2A-594 staining.

[0030] Figure 11Shows the immunocompatibility of HLA-edited HSCs. HLA-edited HSCs and control HSCs (WT, B2M KO, and HLA class II null) were co-cultured with peripheral blood mononuclear cells (PBMCs) that were HLA-B and HLA-C matched but HLA-A mismatched, and PBMC-mediated cytotoxicity was measured by annexin V staining assay.

[0031] Figure 12 Shows the in vivo engraftment potential of HLA-edited HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs (gHSCs) were mixed for competitive transplantation into mice, where bone marrow (BM) and peripheral blood samples were evaluated by FACS to compare the relative amounts of each cell type present in the samples.

[0032] Figure 13A and 13B Shows that the deletion of HLA-A does not affect the presentation of class I peptides. Figure 13A Shows a schematic diagram of immunopeptidome analysis. Figure 13B Shows the results of immunopeptidome analysis, which shows minimal differences in the number of peptides and representative proteins presented by class I molecules between WT and HLA-edited cells.

[0033] Figure 14A and 14B Shows that the deletion of HLA-DP and DQ does not affect the presentation of class II peptides. Figure 14A Shows an immunopeptidome analysis plot. Figure 14B Shows that despite the deletion of HLA-DP and DQ, the cells still retained their ability to present a broad spectrum of peptides via HLA class II.

[0034] Figure 15 Is a schematic diagram of the in vivo testing of antigen-mediated immune responses: the delayed-type hypersensitivity assay (DTH), the sensitization phase, and the elimination phase, respectively.

[0035] Figure 16A and 16B Shows that HLA-edited HSCs reconstituted a functional immune system, as confirmed by the DTH response of immunodeficient mice. Figure 16A Shows the delayed-type hypersensitivity assay performed on transplanted mice, which involves the interaction of different types of immune cells. Mice were sensitized by subcutaneous injection of sheep red blood cells (antigen). A functional immune system led to swelling of the left paw, which was measured with a micrometer caliper. As Figure 16A can be seen, non-transplanted mice did not show any left paw swelling due to their immunodeficiency. In contrast, mice transplanted with cord blood CD34+ cells showed tissue swelling and a doubling of the diameter of their left paw. Figure 16B Is a graphical evaluation of the Figure 16A shown results.

[0036] Figure 17A and 17B showed that WT and HLA-edited HSCs could differentiate into the monocyte / macrophage lineage and also retained the overall expression of both class I and class II molecules, as identified by the CD11b+CD14+ markers ( Figure 17A ). Figure 17B HLA-I and HLA-II analysis of cells gated on CD11b+CD14+ is shown.

[0037] Figures 18A to 18C showed that the absence of HLA-DQB1 and HLA-DPB1 did not affect the expression of other HLA class II molecules. Figure 18A is a schematic diagram of the differentiation of HLA-edited iPSCs into macrophages. Figure 18B is an immunofluorescence experiment that confirmed the specific absence of DPB1 and DQB1 molecules. Figure 18C showed that the same cells retained the expression of class II DRB1.

[0038] The term "gHSC" is used herein to refer to the iPSC-derived hematopoietic stem cells of the present disclosure.

[0039] The terms "wild-type" (WT), "unedited", "non-HLA-edited" are used interchangeably herein to refer to the non-gene-edited cells of the present disclosure.

[0040] EB34+ cells refer to CD34+ cells derived from embryoid bodies. These include hematopoietic endothelial cells. Detailed Description

[0041] The present disclosure provides, in various aspects and embodiments, methods for generating hematopoietic lineages for use in cell therapy, including innate myeloid lineages such as monocytes, macrophages, dendritic cells, and neutrophils, and their precursors. Precursors include common myeloid progenitors (CMPs), granulocyte / macrophage lineage-restricted progenitors (GMPs), macrophage / dendritic cell (DC) progenitors (MDPs), common DC progenitors (CDPs), conventional (or classical) myeloid dendritic cells (cDCs), common monocyte progenitors (cMoPs), and plasmacytoid DCs (pDCs). In various embodiments, the present invention provides efficient in vitro methods for developing such hematopoietic lineages from human induced pluripotent stem cells (iPSCs), including gene-edited iPSCs. The cells generated according to the present disclosure in various embodiments are functional and / or more closely resemble the corresponding lineages isolated from peripheral blood or bone marrow. The present invention also provides isolated cells and cell compositions produced by the methods disclosed herein, as well as methods for cell therapy.

[0042] According to aspects and embodiments of the present disclosure, the ability of human induced pluripotent stem cells (hiPSCs) to generate a substantially unlimited supply of pluripotent stem cells (PSCs) is used to generate an unlimited supply of hematopoietic cells, including but not limited to therapeutic lineages of the innate myeloid lineage that give rise to immune cells or genetically modified versions thereof (e.g., CAR-T expressing cells). The use of the innate immune system myeloid lineage in therapies has been limited by its limited availability, cell numbers, restricted expansion potential, and histocompatibility issues. Additionally, hiPSCs can be more readily genetically modified in vitro compared to primary cells, thus providing improved cell targeting specificity, cell numbers, and bypassing issues such as HLA matching. Moreover, fully engineered hiPSC clones can serve as a stable and safe source compared to primary cells (Nianias and Themeli, 2019). Furthermore, since hiPSCs are of non-embryonic origin, unlike human embryonic stem cells (hESCs), they also have no ethical concerns and are of consistent quality. Thus, using hiPSCs according to the present disclosure has several advantages over primary cells for generating therapeutic hematopoietic lineages such as monocytes, macrophages, dendritic cells (immature and mature dendritic cells), neutrophils, and their precursors.

[0043] In one aspect, the present disclosure provides a method for preparing a cell population comprising myeloid cells in the innate immune system. The method includes preparing a population of pluripotent stem cells (PSCs) (such as a population of induced pluripotent stem cells (iPSCs) differentiated into embryoid bodies), and enriching CD34+ cells to thereby prepare a CD34+-enriched population. Inducing an endothelial-to-hematopoietic cell transition (EHT) in the CD34+-enriched population to thereby prepare a population of hematopoietic stem cells (HSCs), and optionally further enriching CD34+ cells thereafter. The resulting HSC population (or a fraction thereof) can be differentiated into the myeloid lineage of the innate immune system (e.g., phagocytes or their precursors).

[0044] In some aspects and embodiments, the present disclosure provides a method for generating neutrophils, monocytes / macrophages, and immature and mature myeloid DCs (or their precursors) from a population of HSCs.

[0045] Traditionally, hematopoietic lineages have been prepared by differentiating iPSCs into embryoid bodies until day 8 to harvest CD34+ cells. CD34 is commonly used as a marker for hematopoietic endothelial cells, hematopoietic stem cells, and hematopoietic progenitor cells. According to aspects and embodiments of the present disclosure, it has been found that inducing an endothelial-to-hematopoietic cell transition (EHT) in a population of CD34+ cells, which can be derived from iPSC embryoid bodies, can be used to generate advanced hematopoietic lineages in vitro.

[0046] In some embodiments, the myeloid lineage is granulocytes, such as neutrophils. That is, a cell population containing neutrophils differentiates from HSCs. Neutrophils, together with eosinophils and basophils, are subsets of granulocytes. In the event of an attack on the immune system, neutrophils arrive at the scene first. Neutrophil progenitors and mature neutrophils are referred to as proNeu, preNeu, immature Neu, and mature Neu, respectively, which at least partially reflects the conventional morphological classification of myeloblasts, promyelocytes, myelocytes / metamyelocytes, and banded and segmented neutrophils.

[0047] Neutrophils from HSCs can be generated under culture conditions and identified based on the expression of cell surface molecules. For example, different progenitor stages can be defined by the expression of CD117 and CD49d. CD117 mid CD49d high Cells can be classified into SSC low CD34+ cells and SSC high CD34- cells, representing myeloblasts (proNeu1) and promyelocytes (proNeu2 / preNeu), respectively. These cells progress to CD117-CD49d mid and are CD11b+CD101+, which defines myelocytes / metamyelocytes (immature-Neus). CD117-CD49d low cells are CD11b+CD101+ and can additionally express CD16, similar to banded / segmented neutrophils (mature Neu). In addition, these HSC-derived cells gradually express CD35, which is also a marker for the maturation of human myeloid cells in vivo. These cell subsets ((i)CD117 mid CD49d high SSC low CD34+, (ii)CD117 mid CD49d high SSC high CD34-, (iii)CD117-CD49d mid and (iv)CD117-CD49d low ) are morphologically similar to myeloblasts, promyelocytes, myelocytes / metamyelocytes, and neutrophils, respectively.

[0048] By culturing a cell population containing HSCs in the presence of stem cell factor (SCF) and IL-3, promyelocytes can be differentiated from HSCs. In various embodiments, the HSC cell population is further cultured in the presence of granulocyte colony-stimulating factor (G-CSF). By culturing in the presence of G-CSF, promyelocytes can be differentiated into neutrophils.

[0049] In some aspects and embodiments, the myeloid lineage is monocytes or macrophages. Monocytes, macrophages, and dendritic cells are part of the mononuclear phagocyte system in innate immunity, where monocytes are precursors to distinct subsets of macrophages and dendritic cells. They are present in the blood and as resident populations in many organs throughout the body, including the brain, skin, liver, lung, kidney, and heart. They are crucial for controlling pathogens, initiating immune responses, and supporting tissue function.

[0050] By culturing the derived promyelocytes in the presence of G-CSF and M-CSF, the HSC population can be differentiated into CD14+ monocytes, and G-CSF and M-CSF trigger the differentiation of myeloid progenitors into classical monocytes. CD45 expresses a measure of such differentiation. GM-CSF is also added to promote monocyte proliferation. The classical monocyte population is characterized by CD14 and optionally classified by CD11b. Additional cytokines / growth factors, such as but not limited to SCF, TPO, IL-3, and FLT-3 ligand, can supplement GM-CSF to promote robust monocyte generation.

[0051] Phenotypic markers that can be used as monocyte identifiers include, but are not limited to, CD9, CD11b, CD11c, CDw12, CD13, CD15, CDw17, CD31, CD32, CD33, CD35, CD36, CD38, CD43, CD49b, CD49e, CD49f, CD63, CD64, CD65s, CD68, CD84, CD85, CD86, CD87, CD89, CD91, CDw92, CD93, CD98, CD101, CD102, CD111, CD112, CD115, CD116, CD119, CDw121b, CDw123, CD127, CDw128, CDw131, CD147, CD155, CD156a, CD157, CD162, CD163, CD164, CD168, CD171, CD172a, CD180, CD206, CD131a1, CD2132, CDw210, CD226, CD281, CD282, CD284, and CD286. In certain embodiments, monocytes include CD14+CD16− monocytes, CD14+CD16+ monocytes, or CD14−CD16+ monocytes. In various embodiments, neutrophils release myeloperoxidase (MPO), which is a key element of the innate immune system, to provide defense against invading pathogens. Exposure of neutrophils to inflammatory mediators (e.g., chemokines, cytokines, complement proteins, or oxidants such as HOCl) also triggers the release of neutrophil extracellular traps (NETs). Thus, in some embodiments, MPO is used as a marker to measure neutrophil activation.

[0052] In some embodiments, monocytes differentiate into macrophages. For macrophage differentiation, CD14+ cells are cultured in the presence of human M-CSF. Protocols for differentiating monocytes into macrophages are well known to those skilled in the art.

[0053] In some embodiments, cells differentiate into dendritic cells (DCs). For DC differentiation, CD14+ cells are supplemented with GM-CSF and IL-4. In some embodiments, the cells are seeded under ultra-low attachment culture conditions and allowed to further differentiate into dendritic cells. Maturation of dendritic cells can be achieved by supplementing the medium with LPS and / or TNFα. Other factors that can be supplemented include IL-1β, INF-γ, and PGE-2. Protocols for differentiating monocytes into DCs are well known to those skilled in the art. According to the present disclosure, various types of macrophage populations can be generated in vitro.

[0054] Macrophages are distributed in various tissues and organs throughout the body and exhibit a high degree of heterogeneity and diversity. Several specific markers expressed on the surface of macrophages have been used to identify different subsets, such as F4 / 80, CD68, SRA-1, and CD169(2). CD169+ macrophages are a unique subset of macrophages distributed in multiple tissues and organs of the human body.

[0055] Macrophages derived from monocyte precursors undergo specific differentiation depending on the local tissue environment. They respond to environmental factors within the tissue (such as damaged cells, activated lymphocytes, or microbial products) and differentiate into different functional phenotypes. The M1 macrophage phenotype is characterized by the production of high levels of pro-inflammatory cytokines, the ability to mediate resistance to pathogens, strong bactericidal properties, the production of large amounts of reactive nitrogen and oxygen intermediates, and the promotion of Th1 responses. In contrast, M2 macrophages are characterized by their involvement in parasite control, tissue remodeling, immune regulation, tumor promotion, and efficient phagocytic activity. M2 macrophages can be further divided into multiple subsets based on their different gene expression profiles, specifically M2a, M2b, M2c, and M2d.

[0056] Commonly expressed M1 macrophage markers include but are not limited to: CD64, IDO, SOCS1, CXCL10, CD86, CD80, MHCII, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, PD-L1, CD69, MHCI, CD32, CD16, IFIT family members, or ISG family members; while commonly expressed human M2 macrophage markers include but are not limited to multifunctional enzyme transglutaminase 2 (TGM2), MRC1, CD23, CCL22, CD206, CD163, and / or CD209.

[0057] Macrophages also include T cell receptor+ and CD169+ macrophages. These macrophages express the TCR co-receptor CD3 as well as the TCRαβ and γδ subtypes. TNF is one of the key regulators of TCRαβ expression in macrophages. TCRγδ macrophages are involved in the host defense against bacterial challenge. Both TCR+ macrophage subsets express molecules that have been shown to be essential for T cell signaling, such as ZAP70, LAT, Fyn, and Lck. In addition, they exhibit strong phagocytic ability and secrete the chemokine CCL2.

[0058] CD169+ macrophages are mainly located in secondary lymphoid organs but redistribute after immune activation. CD169+ macrophages are able to present antigens to B cells and activate CD8+ T cells. CD169+ macrophages are involved in immune tolerance induced by apoptotic cell clearance and play anti-tumor and anti-viral roles.

[0059] In some embodiments, the myeloid lineage is the dendritic cell (DC) lineage. DCs detect homeostatic imbalances and process antigens for presentation to T cells, thereby establishing a link between innate and adaptive immune responses. In addition, DCs can secrete cytokines and growth factors to regulate ongoing immune responses and are influenced by their interactions with other immune cells such as natural killer cells and innate lymphoid cells (ILCs).

[0060] DCs have two distinct functional states: "mature" and "immature". These are distinguished by multiple characteristics, but activation of naive antigen-specific T cells in secondary lymphoid organs is a hallmark of mature DCs. DC maturation is triggered by tissue homeostatic dysregulation and detected by recognition of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs).

[0061] Identifiers of DCs include but are not limited to CD45+CD11c+CD1c+ and HLA-DR+. After LPS stimulation, DCs undergo a maturation process and upregulate co-stimulatory molecules such as CD80 and CD40, while CD16, HLA-DR, and PDL1 are unaffected. Other phenotypic markers that can be used as DC identifiers include but are not limited to CD83, CD1a, CD1c, CD141, CD207, CLEC9a, CD123, CD85, CD180, CD187, CD205, CD281, CD282, CD284, CD286, and parts of CD206, CD207, CD208, and CD209.

[0062] DCs can be further divided into multiple subtypes such as plasmacytoid DCs (pDCs) (fcer1 / ILT3 / ILT7 / DR6), myeloid / conventional DC1 (cDC1) (CD141 / CLEC9A / XCR1 / CADM1 / BTLA), and myeloid / conventional DC2 (cDC2) (CD1c / CD172a / FcεR1 / SIRPA) and LCs (langerin / CD1a).

[0063] In various embodiments, granulocytes or phagocytes or their progenitors (as described herein) are generated by contacting CD34+ cells (e.g., recovered from EB dissociation) with an effective amount of a mechanosensitive receptor or a mechanosensitive channel agonist that increases the activity or expression of Dnmt3b. In various embodiments, the CD34+ cells are further cultured in a medium comprising one or more growth factors and cytokines selected from TPO, SCF, Flt3L, IL3, IL-6, IL7, IL-11, IGF, bFGF, and IL15, which medium optionally comprises one or more of VEGF, bFGF, BMP activator, Wnt pathway activator, or ROCK inhibitor (e.g., thiazovivin or Y27632). The resulting HSCs can be cultured in the presence of growth factors and cytokines such as, but not limited to, IL-3, IL-7, IL-15, SCF, and FLT-3L. The cells can be cultured in the presence of M-CSF and / or G-CSF to differentiate into the myeloid lineage (as described above) and optionally supplemented with IL-4 and TNF-α (as described).

[0064] In some embodiments, CD34+ cells (i.e., recovered from EB dissociation) are contacted with an effective amount of a mechanosensitive receptor or a mechanosensitive channel agonist that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensitive receptor is Piezol. Exemplary Piezol agonists include Yoda1, single-stranded (ss) RNA (e.g., ssRNA40), Jedi1, and Jedi2 or analogs thereof. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. The manner of inducing EHT (whether or not a mechanosensitive receptor agonist is used) can be employed and is described herein. In some embodiments, after inducing EHT, the cells (HSCs or their progeny) differentiate into the innate myeloid lineage of immune cells, i.e., the phagocyte lineage.

[0065] In various embodiments, iPSCs are prepared by reprogramming somatic cells. The term "induced pluripotent stem cell" or "iPSC" refers to a cell derived from a somatic cell, such as a skin or blood cell that has been reprogrammed back to an embryonic-like pluripotent state. In some embodiments, iPSCs are generated from somatic cells such as (but not limited to) fibroblasts or PBMCs (or cells isolated therefrom). In some embodiments, iPSCs are derived from lymphocytes, granulocyte / macrophage lineage-restricted progenitors (GMPs), umbilical cord blood cells, PBMCs, CD34+ cells, or other human primary tissues. In some embodiments, iPSCs are derived from CD34+ cells isolated from peripheral blood. In various embodiments, the iPSCs are autologous or allogeneic (e.g., HLA-matched at one or more loci) to the recipient (the subject in need of treatment as described herein). In various embodiments, the iPSCs can be gene-edited to facilitate HLA matching (such as deleting one or more HLA class I and / or class II alleles or their master regulators, including but not limited to β-2-microglobulin (B2M), CIITA, etc.), or gene-edited to cause other loss-of-function or express other functions. For example, the iPSCs can be gene-edited to delete one or more of HLA-A, HLA-B, and HLA-C, and one or more of HLA-DP, HLA-DQ, and HLA-DR. In certain embodiments, the iPSCs retain the expression of at least one HLA class I and at least one HLA class II complex. In certain embodiments, the iPSCs are homozygous for at least one retained class I and class II locus.

[0066] In various embodiments, the iPSCs are gene-edited to be one of the following: (i) HLA-A-B+C+DP-DR+DQ+, (ii) HLA-A-B+C+DP+DR+DQ-, (iii) HLA-A-B+C+DP-DR+DQ-; (iv) HLA-A-B-C+DP-DR+DQ+; (v) HLA-A-B-C+DP+DR+DQ-, (vi) HLA-A-B-C+DP-DR+DQ-. For the retained HLAs (e.g., HLA-B, HLA-C, and HLA-DR), the cells can be homozygous or retain only a single copy of the gene. For example, the modified cells are at least identified as (a) HLA-C+ and HLA-DR+, and optionally identified as one or more of (b) HLA-B-, (c) HLA-DP-, and (d) HLA-DQ-. In an exemplary embodiment, the modified cells are HLA-B+, HLA-DP-, and HLA-DQ-.

[0067] In some embodiments, the iPSCs are gene-edited to HLA-A neg, for those that are homozygous for both HLA - B and HLA - C, as well as HLA - DPB1 neg and HLA - DQB1 neg . In some embodiments, the iPSCs are further homozygous for HLA - DRB1.

[0068] As used herein, the term "negative" (-) or "negative" with respect to a particular HLA class I or class II molecule indicates that both copies of the gene have been disrupted in a cell line or population, and thus the cell line or population does not display significant functional expression of the gene. Such cells can be generated by complete or partial gene deletion or disruption, or alternatively by other techniques such as siRNA. As used herein, the term "deletion" in the context of genetic modification (i.e., gene editing) of a target gene refers to the abolition of the functional expression of the corresponding gene product (i.e., the corresponding polypeptide). Such gene editing includes complete or partial gene deletion or disruption of the coding sequence, or deletion of key cis - acting expression control sequences.

[0069] Somatic cells can be reprogrammed by expressing reprogramming factors selected from Sox2, Oct3 / 4, c - Myc, Nanog, Lin28, and Klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c - Myc, Nanog, Lin28, and Klf4. In some embodiments, the reprogramming factors are Sox2, Oct3 / 4, c - Myc, and Klf4. Methods for preparing iPSCs are described, for example, in U.S. Patent 10,676,165; U.S. Patent 9,580,689; and U.S. Patent 9,376,664, which are hereby incorporated by reference in their entireties. In various embodiments, well - known viral vector systems such as lentivirus, Sendai virus, or measles virus systems are used to express the reprogramming factors. Alternatively, the reprogramming factors can be expressed by introducing mRNA encoding the reprogramming factors into somatic cells. In addition, iPSCs can be generated by introducing non - integrating episomal plasmids that express the reprogramming factors, i.e., for generating transgene - free and virus - free iPSCs. Known episomal plasmids can be employed, which have limited replication ability and thus will be lost after several generations of cells.

[0070] In some embodiments, human pluripotent stem cells (e.g., iPSCs) are gene-edited. Gene editing can include, but is not limited to, for example, HLA gene modification (e.g., deletion of one or more HLA class I and / or class II genes), deletion of β2-microglobulin (β2M), deletion of CIITA, deletion or addition of granulocyte or phagocyte receptor genes, or addition of a chimeric antigen receptor (CAR) gene. Exemplary CAR cells (e.g., monocytes, macrophages, dendritic cells, and neutrophils) can target tumor antigens, such as one or more of CD19, CD38, CD33, CD47, and CD20.

[0071] In some embodiments, iPSCs are gene-edited using gRNAs that are 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more nucleotides in length. In some embodiments, the gRNA comprises a modification at or near the 5'-end (e.g., within 1 to 10, 1 to 5, or 1 to 2 nucleotides of the 5'-end) and / or a modification at or near the 3'-end (e.g., within 1 to 10, 1 to 5, or 1 to 2 nucleotides of the 3'-end). In some embodiments, the modified gRNA exhibits increased resistance to nucleases. In some embodiments, the gRNA comprises two separate RNA molecules (i.e., a "dual gRNA"). The dual gRNA comprises two separate RNA molecules: "crispr RNA" (or "crRNA") and "tracr RNA", and is well known to those skilled in the art.

[0072] Generally, various gene editing techniques are known and can be applied in accordance with various embodiments of the present disclosure. Gene editing techniques include, but are not limited to, zinc fingers (ZFs), transcription activator-like effectors (TALEs), and the like. Fusion proteins containing one or more of these DNA binding domains and the cleavage domain of the Fokl endonuclease can be used to generate double-strand breaks in the desired region of DNA in a cell (see, for example, U.S. Patent Application Publication No. US2012 / 0064620, U.S. Patent Application Publication No. US2011 / 0239315, U.S. Patent No. 8,470,973, U.S. Patent Application Publication No. US 2013 / 0217119, U.S. Patent No. 8,420,782, U.S. Patent Application Publication No. US2011 / 0301073, U.S. Patent Application Publication No. US2011 / 0145940, U.S. Patent No. 8,450,471, U.S. Patent No. 8,440,431, U.S. Patent No. 8,440,432, and U.S. Patent Application Publication No. 2013 / 0122581, the entire contents of each of which are hereby incorporated by reference). In some embodiments, gene editing is performed using a CRISPR-associated Cas system known in the art (e.g., CRISPR-Cas9). See, for example, US 8,697,359, US 8,906,616, and US 8,999,641, each of which is hereby incorporated by reference in its entirety. In various embodiments, gene editing employs a type II Cas endonuclease (such as Cas9) or a type V Cas endonuclease (such as Cas12a). Type II and type V Cas endonucleases are RNA-guided. gRNA design for guiding desired gene editing (while limiting or avoiding off-target editing) is known in the art. See, for example, Mohr SE et al., CRISPR guideRNA design for research applications ,FEBS J. September 2016; 283(17):3232–3238. In other embodiments, non-canonical type II or type V Cas endonucleases that are homologous (although with low primary sequence homology) to Streptococcus pyogenes Cas9 or Prevotella and Francisella 1 (Cpf1 or Cas12a) can be employed. Many such non-canonical Cas endonucleases are known in the art. Nidhi S, et al. Novel CRISPR–Cas Systems: An Updated Review of theCurrent Achievements,Applications, and Future Research Perspectives ,Int J MolSci. April 2021; 22(7):3327. In other embodiments, gene editing employs base editing or prime editing to incorporate mutations without causing double-strand breaks. See, for example, Antoniou P et al.,Base and Prime Editing Technologies for Blood Disorders , Front. Genome Ed., January 28, 2021; Matsuokas IG, Prime Editing: Genome Editing for Rare Genetic Diseases Without Double - Strand Breaks or Donor DNA , Front. Genet., June 9, 2020. Various other gene editing processes are known, including using a dead Cas (dCas) system (e.g., Cas fusion protein) to direct a DNA modifying enzyme to a desired target, using dCas as a guide RNA-directed system. Brezgin S, Dead Cas Systems: Types, Principles, and Applications , Int. J. Mol. Sci. December 2019; 20(23):6041.

[0073] Base editors that can install precise genomic alterations without generating double-strand DNA breaks can also be used for gene editing in cells (e.g., iPSCs) (e.g., engineering gene therapy vectors). Base editors essentially comprise a catalytically disabled nuclease, such as Cas9 nickase (nCas9), which cannot generate DSBs, and are fused to a nucleobase deaminase and, in some cases, a DNA glycosylase inhibitor. Currently, there are 2 main types of base editors, cytosine base editors (CBEs) and adenine base editors (ABEs), which catalyze C>T and A>G conversions. Base editors can be delivered, for example, via an HDAd5 / 35++ vector to efficiently edit promoters and enhancers to activate or inactivate genes. Exemplary methods are described in U.S. Patent Nos. 9,840,699; 10,167,457; 10,113,163; 11,306,324; 11,268,082; 11,319,532; and 11,155,803. Primer editors, which comprise a reverse transcriptase conjugated (e.g., fused) to a Cas endonuclease and a polynucleotide conjugated (e.g., fused) to a guide RNA that serves as a DNA synthesis template, are also envisioned, as described in WO 2020 / 191153.

[0074] Exemplary vectors useful for genome editing applications include, but are not limited to, plasmids, retroviral vectors, lentiviral vectors, adenoviral vectors (e.g., Ad5 / 35, Ad5, Ad26, Ad34, Ad35, Ad48), parvoviruses (e.g., adeno-associated virus (AAV) vectors, herpes simplex virus vectors, baculovirus vectors, coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses (including herpesviruses (e.g., herpes simplex virus type 1 and 2, Epstein-Barr virus, cytomegalovirus) and poxviruses (e.g., canarypox virus, vaccinia virus, or modified vaccinia virus)). A vector comprising a nucleic acid molecule of interest can be delivered to a cell (e.g., iPS cells, endothelial cells, hematopoietic endothelial cells, HSCs (ST-HSCs or LT-HSCs)) by any method known in the art, including but not limited to transduction, transfection, infection, and electroporation. Any of these vectors can include transposable elements (such as piggyBac transposons or Sleeping Beauty transposons). Transposons insert specific DNA sequences into the genome of vertebrates. Once excised from the transposon, the gene can be integrated into the genome of mammalian cells by catalytic cleavage of a similar excision site present in the nuclear genome by a transposase.

[0075] To increase efficiency, in some embodiments, Cas and gRNA can be combined before being delivered to the cell. The Cas-gRNA complex is referred to as a ribonucleoprotein (RNP). Many methods have been developed to directly deliver RNPs to cells. For example, RNPs can be delivered to cells in culture by lipofection or electroporation. Electroporation using a nucleofection protocol can be employed, and this procedure allows rapid entry of the RNP into the nucleus, so genome cleavage can begin immediately. See, e.g., Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleopro tein for CRISPR / Cas9 genome editing .Theranostics. January 1, 2021; 11(2):614-648, which is hereby incorporated by reference in its entirety. In some embodiments, Cas9 and gRNA are electroporated into donor iPSCs and / or HSCs as RNPs.

[0076] Typically, a protospacer adjacent motif (PAM) is required for Cas nuclease cleavage, and the protospacer adjacent motif is typically found 3 to 4 nucleotides downstream of the cleavage site. The PAM is a short DNA sequence (usually 2 to 6 base pairs in length) that lies after the DNA region targeted for cleavage by a CRISPR system such as CRISPR-Cas9. In some embodiments, the PAM sequence, sgRNA, or base editing tool targeting a haplotype or polymorphism of the HLA locus does not include four Gs, four Cs, GC repeat sequences, or combinations thereof.

[0077] In some embodiments, a CRISPR / Cas9 system specific for a unique HLA haplotype can be developed using the gRNAs described herein by designing a single gRNA targeting each of the donor-specific HLA-A, HLA-DPB1, and HLA-DQB1 genes (for example). For gene knockout, the gRNA targets the Cas9 protein to a suitable site for editing. Next, the Cas9 protein can perform a double-strand break (DSB), where the DNA is repaired by the non-homologous end joining (NHEJ) mechanism, which generates indels that result in frameshift mutations and terminate the function of the resulting protein. However, off-target gene modification can occur and alter the function of other intact genes. For example, even in the presence of a certain degree of mismatch, the Cas9 endonuclease can generate DSBs at unwanted off-target locations. Such off-target activity can produce genomic instability events such as point mutations and genomic structural variations. In various embodiments, the sgRNA targeting HLA-A can target a region of chromosome 6 defined as 29942532-29942626. In various embodiments, the sgRNA targeting HLA-DQB1 can target a region of chromosome 6 defined as 32665067-32664798. In various embodiments, the sgRNA targeting HLA-DPB1 can target a region of chromosome 6 defined as 33080672-33080935.

[0078] gRNAs can be used to develop cloned iPSCs. (i) On-target editing, (ii) off-target editing, and (iii) translocation editing of such iPSC lines can be evaluated, for example, using sequencing as described herein. Specifically, such assays can be performed by multiplex PCR that utilizes primers designed to target and enrich regions of interest, followed by next-generation sequencing (e.g., amplicon sequencing, AMP-seq). The on-target and translocation panels can amplify the expected edited regions, allowing selection of iPSC clones with the expected edits that do not have chromosomal translocations caused by fusion of unintended DSB cleavage sites. The off-target panel can enrich any potential off-target regions identified by sequencing and allow selection of iPSC clones with negligible off-target mutations. In summary, these assays enable screening of iPSC clones to select clones with the desired edits while excluding potential CRISPR / Cas9-related genomic integrity issues.

[0079] In some embodiments, to further ensure the genomic stability and integrity of reprogrammed and edited iPSCs, genetic and genomic assays can be performed to select clones, for example, that have not undergone translocation and mutation events and that have not integrated episomal vectors. For example, whole-genome sequencing (WGS) is performed on CD34+ cells and the reprogrammed iPSC clones, where genomic differences resulting from editing are compared. These analyses provide an assessment of which iPSC clone genomes differ from the CD34+ starting material, enabling informed selection of iPSC clones that do not generate mutations during reprogramming.

[0080] In some embodiments, karyotyping using a system such as the KARYOSTAT assay is used to select iPSC clones that do not generate indels and translocations during reprogramming, for example, as described in Ramme AP, et al., Supporting data set of two integration - free induced pluripotent stem cell lines from related human donors ,”Data Brief. May 15, 2021; 37:107140, which is hereby incorporated by reference in its entirety. The KARYOSTAT assay allows visualization of chromosomal aberrations at a resolution similar to that of G-banded karyotyping. For chromosomal gains, the size of the structural aberrations that can be detected >2 Mb, and for chromosomal losses, the size of the structural aberrations that can be detected >1 Mb. The KARYOSTAT array is functionalized to achieve balanced whole-genome coverage by low-resolution DNA copy number analysis, where the assay covers all 36,000 RefSeq genes, including 14,000 OMIM targets. The assay is capable of detecting aneuploidy, submicroscopic aberrations, and mosaic events.

[0081] In some embodiments, array comparative genomic hybridization (aCGH) analysis is used to select iPSC clones that do not generate copy number aberrations (CNA) during reprogramming, such as as described in Wiesner et al. “ Molecular Techniques ”, edited by Klaus J. Busam, Pedram Gerami, Richard A. Scolyer, “Pathology of Melanocytic Tumors,” Elsevier, 2019, pages 364 - 373, ISBN 9780323374576; and Hussein SM, et al. “Copy number variation and selection during reprogramming to pluripotency,” Nature. March 3, 2011; 471(7336):58 - 62, which are hereby incorporated by reference in their entirety. aCGH is a technique for analyzing the entire genome for CNA by comparing sample DNA and reference DNA.

[0082] In some embodiments, targeted hematopoietic malignancy NGS panel analysis is used to select iPSC clones that do not generate hematopoietic malignancy mutations during reprogramming. For example, the targeted hematopoietic malignancy NGS panel can focus on myeloid leukemia, lymphoma, and / or other genes associated with hematopoietic malignancies to generate a smaller, more manageable dataset than a more extensive approach. Targeted hematopoietic malignancy NGS panel analysis includes using highly multiplexed PCR to amplify regions associated with hematopoietic malignancies, followed by next - generation sequencing.

[0083] In some embodiments, droplet digital PCR (ddPCR) is used to select iPSC clones that do not integrate episomal vectors and have been passaged sufficiently for episomal vector clearance. As described herein, iPSC reprogramming of CD34+ cells can be achieved by delivery of episomal vectors encoding reprogramming factors. However, although rare, episomal vectors can randomly integrate into the cell genome, which may disrupt developmental processes, homeostasis, etc. Thus, the ddPCR method can be used to detect residual episomal vectors in iPSC cultures and is capable of selecting iPSC clones that do not integrate episomal vectors.

[0084] In some embodiments, after evaluating that the selected clone has no genome aberrations associated with editing, the clone can be additionally tested for spontaneous mutations that may occur during expansion. For example, mutations affecting hematologic malignancy genes, indels, translocations, numerical aberrations, such as those described for pre-edited reprogrammed clones. Analysis of spontaneous mutations can include whole genome sequencing (WGS), KARYOSTAT analysis, array comparative genomic hybridization (aCGH) analysis, targeted hematologic malignancy NGS panel AMP-Seq analysis, and / or droplet digital PCR (ddPCR).

[0085] In various embodiments, iPSCs are prepared and expanded using a culture system. The expanded iPSCs can be recovered from the culture to generate embryoid bodies (EBs). EBs differentiated from iPSCs are three-dimensional aggregates of iPSCs and contain three (or two or one) embryonic germ layers based on the differentiation method. For example, the preparation of EBs is described in US2019 / 0177695, which is hereby incorporated by reference in its entirety. In some embodiments, EBs prepared by differentiation of iPSCs are expanded in a bioreactor, such as described by Abecasis B. et al., Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling - up approaches .J. of Biotechnol. 246 (2017) 81-93. EBs can be used to generate any desired cell type. Other methods for the expansion or differentiation of EBs, including 3D suspension culture, are described in WO 2020 / 086889, which is hereby incorporated by reference in its entirety.

[0086] In some embodiments, the methods according to each aspect can include generating CD34+-enriched cells from pluripotent stem cells (e.g., EBs) and inducing endothelial cell to hematopoietic cell differentiation. HSCs containing a relatively high frequency of LT-HSCs can be generated from a cell population using: various stimuli or factors, including mechanical, biochemical, metabolic, and / or topographical stimuli, and factors such as induction of extracellular matrix, niche factors, extracellular extrinsic factors, cell-intrinsic properties; and including pharmacological and / or genetic means.

[0087] In some embodiments, the method includes preparing endothelial cells with hematopoietic potential from pluripotent stem cells prior to EHT induction. In some embodiments, overexpression of combinations of GATA2 / ETV2, GATA2 / TAL1, or ER71 / GATA2 / SCL can result in the formation of endothelial cells with hematopoietic potential from PSC sources. In some embodiments, the method includes overexpressing the E26 transformation-specific variant 2 (ETV2) transcription factor in iPSCs. ETV2 can be expressed by introducing a plasmid encoding a non-integrating episome for constitutive or inducible expression of ETV2 and for generating transgene-free hematopoietic ECs. In some embodiments, ETV2 is expressed from mRNA introduced into iPSCs. Any available method can be used to introduce the mRNA, including electroporation or lipofection. Differentiation of cells expressing ETV2 can include addition of VEGF-A. See Wang K, et al., Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with mRNA .Sci. Adv. Vol. 6 (2020). According to embodiments of the present disclosure, cells generated in this manner can be used to generate CD34+ cells and induce EHT.

[0088] After CD34+ enrichment, HSCs are generated from endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimulation or modification.

[0089] In some embodiments, iPSC differentiation proceeds until the cells are at least about 10% CD34+, or at least about 20% CD34+, or at least about 25% CD34+, or at least about 30% CD34+. In some embodiments, CD34 enrichment and EHT can be induced on days 7 to 14 (such as, for example, day 8, day 9, day 10, day 11, day 12, day 13, or day 14) of iPSC differentiation. Differentiation of iPSCs can be performed according to known techniques. In some embodiments, iPSC differentiation involves combinations of factors such as, but not limited to, bFGF, Y27632, BMP4, VEGF, SCF, EPO, TPO, IL-6, IL-11, and / or IGF-1. In some embodiments, feeder-free, serum-free, and / or GMP-compatible materials are used to differentiate hPSCs. Serum-free cultures typically contain a mixture of cytokines / growth factors / small molecules.

[0090] Monocyte lineage differentiation can be performed in 5 consecutive steps as disclosed below: Yanagimachi et al. Robustand Highly Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent Stem Cells under Serum - and Feeder Cell - Free Conditions .PLOS ONE. April 3, 2013; 8(4): e59243 or its improvement, such as but not limited to replacing or omitting one of the cytokines or steps to accelerate the differentiation of functional monocytes.

[0091] In a non-limiting example, isolated monocytes can be cultured in serum-free medium supplemented with M-CSF. Then, M1 macrophages can be stimulated by adding LPS and IFN-γ, or M2a macrophage polarization can be stimulated by adding IL-4. M1 macrophages can be identified by high levels of CD80 and CCR7 markers and low levels of CD206 and CD209. In contrast, M2a macrophages can be identified by CD206 and CD209, which are optionally negative or at low levels for CD80 and CCR7. The functionality of macrophages can be demonstrated by well-known techniques, such as but not limited to phagocytosis assays using fluorescently labeled Escherichia coli.

[0092] Alternatively, in some embodiments, feeder cells (such as STO mouse fibroblast feeder layers) can be used to expand myeloid cells (e.g., phagocytes or their precursors). In some embodiments, hPSCs are co-cultured with feeder layers of murine bone marrow-derived feeder cells such as OP9, STO mouse fibroblasts, or blood-derived peripheral blood mononuclear cells (PBMCs), or umbilical cord blood-derived mesenchymal stem cells, or lymphocyte-derived cancer cell line cells in serum-containing medium. The culture can contain growth factors and cytokines to support the differentiation of embryoid bodies or monolayer systems. The feeder cell co-culture system can be used to generate pluripotent HSPCs, which can further differentiate into several hematopoietic lineages, including monocytes or macrophages, dendritic cells, neutrophils, NK cells, T lymphocytes, B lymphocytes, megakaryocytes, and erythrocytes. See Netsrithong R. et al., Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells , Stem Cell Research & Therapy Vol. 11 Art. 481 (2020). Alternatively, a stepwise process using defined conditions with specific signals can be used. For example, the expression of HOXA9, ERG, RORA, SOX4, and MYB in human PSCs favors direct differentiation into CD34+ / CD45+ progenitor cells with multilineage potential. In addition, the expression of factors such as HOXB4, CDX4, SCL / TAL1, or RUNX1a supports the hematopoietic program in human PSCs. See Doulatov S. et al., Induction ofmultipotential hematopoietic progenitorsfromhumanpluripotent stemcells via re-specification oflineage-restricted precursors ,Cell Stem Cell. October 3, 2013; 13(4).

[0093] The induction of EHT can be carried out by any known method. In some embodiments, the induction of EHT generates a population of hematopoietic stem cells (HSCs) that includes LT-HSCs. In some embodiments, EHT generates HSCs from endothelial cells or hematopoietic endothelial cell (HEC) precursors using mechanical, biochemical, pharmacological, and / or genetic means (e.g., by stimulation, inhibition, and / or genetic modification). In some embodiments, EHT generates a stem cell population that includes one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs), and hematopoietic stem cell progenitors. In an embodiment, EHT is carried out using a culture medium that includes one or more growth factors and cytokines selected from TPO, SCF, Flt3L, IL3, IL-6, IL7, IL-11, IGF, bFGF, and IL15. The culture medium can optionally include one or more of VEGF, bFGF, BMP activator, Wnt pathway activator, or ROCK inhibitor (e.g., thiazovivin or Y27632).

[0094] In some embodiments, the method includes increasing the expression or activity of dnmt3b in PSCs, embryoid bodies, CD34+-enriched cells, ECs, HECs, or HSCs, which can be carried out by mechanical, genetic, biochemical, or pharmacological means. In some embodiments, the method includes increasing the activity or expression of DNA (cytosine-5-)-methyltransferase 3β (Dnmt3b) and / or GTPase IMAP family member 6 (Gimap6) in the cells. See WO 2019 / 236943 and WO 2021 / 119061, which are hereby incorporated by reference in their entireties. In some embodiments, the induction of EHT includes increasing the expression or activity of dnmt3b.

[0095] In some embodiments, the cells are contacted with an effective amount of a mechanosensor or a mechanosensitive channel agonist that increases the activity or expression of Dnmt3b. In some embodiments, the mechanosensor is Piezol. An exemplary Piezol agonist is Yoda1. In some embodiments, the mechanosensor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. Yodal (2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine) is a small molecule agonist developed against the mechanosensitive ion channel Piezol. Syeda R, Chemical activation ofthe mechanotransduction channel Piezol .eLife(2015).

[0096] Derivatives of Yodal can be used in various embodiments. For example, in some embodiments, derivatives containing a 2,6-dichlorophenyl nucleus are employed. Exemplary agonists are disclosed in Evans EL, et al., Yoda1 analogue (Dooku1) which antagonizesYoda1-evoked activation ofPiezo1 and aortic relaxation ,British J. of Pharmacology 175(1744 - 1759):2018. Other Piezo1 agonists include Jedi1, Jedi2, ssRNA40, and their derivatives and analogs. See Wang Y., et al., Alever-like transduction pathwayfor long-distancechemical- and mechano-gatingofthemechanosensitivePie zo1 channel .Nature Communications(2018)9:1300; Sugisawa et al., RNASensingby Gut Piezo1 Is Essential for Systemic Serotonin Synthesis ,Cell, Volume 182, Issue 3, 2020, Pages 609 - 624, which is incorporated herein by reference in its entirety. These Piezo1 agonists are commercially available. In various embodiments, an effective amount of a Piezo1 agonist or derivative is in the range of about 1 μM to about 500 μM, or about 5 μM to about 200 μM, or about 5 μM to about 100 μM, or in some embodiments, in the range of about 25 μM to about 150 μM, or about 25 μM to about 100 μM, or about 25 μM to about 50 μM.

[0097] In various embodiments, pharmacological Piezo1 activation is applied to CD34+ cells (i.e., CD34+-enriched cells). In certain embodiments, pharmacological Piezo1 activation can be further applied to iPSCs, embryoid bodies, ECs, hematopoietic endothelial cells (HECs), HSCs, hematopoietic progenitor cells, and hematopoietic lineages. In certain embodiments, Piezo1 activation is applied at least to EBs generated from iPSCs, CD34+ cells isolated from EBs, and / or combinations thereof.

[0098] Alternatively or additionally, the activity or expression of Dnmt3b can be directly increased in cells (e.g., in CD34-enriched cells). For example, the mRNA expression of Dnmt3b can be increased by delivering transcripts encoding Dnmt3b to the cells, or by introducing a transgene encoding Dnmt3b, or by non-transgenic methods (not limited to introducing non-integrating episomes into the cells). In some embodiments, gene editing is employed to introduce genetic modifications into the Dnmt3b expression element in the cells, such as but not limited to increasing promoter strength, ribosome binding, RNA stability, and / or affecting RNA splicing.

[0099] In some embodiments, the method includes increasing the activity or expression of Gimap6 in the cells, alone or in combination with Dnmt3b and / or other genes that are upregulated or downregulated upon cyclic strain or piezoelectric activation. To increase the activity or expression of Gimap6, the mRNA transcript encoding Gimap6 can be introduced into the cells, or non-transgenic methods can be employed, including but not limited to introducing episomes into the cells; or alternatively a transgene encoding Gimap6. In some embodiments, gene editing is used to introduce genetic modifications into the Gimap6 expression element in the cells (such as one or more modifications to increase promoter strength, ribosome binding, RNA stability, or affecting RNA splicing).

[0100] In embodiments of the present disclosure that employ mRNA delivery to the cells, known chemical modifications can be used to avoid the innate immune response in the cells. For example, synthetic RNA containing only canonical nucleotides can bind to pattern recognition receptors and can trigger an effective immune response in the cells. Such a response can lead to translation blockage, secretion of inflammatory cytokines, and cell death. RNA containing certain non-canonical nucleotides can evade detection by the innate immune system and can be efficiently translated into proteins. See US 9,181,319, which is hereby incorporated by reference, particularly with respect to nucleotide modifications to avoid the innate immune response.

[0101] In some embodiments, the expression of Dnmt3b and / or Gimap6 is increased by introducing a transgene into the cells, which can direct the desired overexpression level (with other options of different promoter strengths or expression control elements). Various viral vectors or transfection reagents known in the art (including lipid nanoparticles) can be used to introduce the transgene. In some embodiments, the expression of Dnmt3b and / or Gimap6 is increased by non-transgenic methods (e.g., episomal delivery). In some embodiments, gene editing techniques are used to increase the expression or activity of Dnmt3b and / or Gimap6 or other genes disclosed herein, e.g., to introduce one or more modifications to increase promoter strength, ribosome binding, or RNA stability.

[0102] In some embodiments, the method does not involve increasing the expression of dnmt3b, such as by using a Piezo1 agonist.

[0103] In some embodiments, the method includes applying cyclic 2D, 3D, or 4D stretch to the cells. In various embodiments, the cells subjected to cyclic 2D, 3D, or 4D stretch are selected from one or more of CD34-enriched cells, iPSCs, ECs, and HECs. For example, a cell population is introduced into a bioreactor that provides cyclic strain biomechanical stretch, as described in WO 2017 / 096215, which is hereby incorporated by reference in its entirety. The cyclic strain biomechanical stretch can increase the activity or expression of Dnmt3b and / or Gimap6. In these embodiments, mechanical means apply a stretching force to the cells or to the cell culture surface on which the cells (e.g., ECs or HECs) are cultured. For example, a computer-controlled vacuum pump system or other components for providing a stretching force attached to a flexible biocompatible and / or biomimetic surface (e.g., FlexCell TM Tension System, Cytostretcher System) can be used to apply in vitro cyclic 2D, 3D, or 4D stretch to the cells under defined and controlled cyclic strain conditions. For example, the cyclic stretch applied can be a cyclic strain of about 1% to about 20% (e.g., about 6% cyclic strain) for several hours or days (e.g., about 7 days). In various embodiments, the cyclic strain is applied for at least about one hour, at least about two hours, at least about six hours, at least about eight hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, or at least about 168 hours. In various embodiments, no cyclic stretch is employed.

[0104] Alternatively or additionally, EHT is stimulated through Trpv4 activation. Trpv4 activation can be achieved by contacting the cells (e.g., CD34-enriched cells, ECs, or HECs) with one or more Trpv4 agonists, which are optionally selected from GSK1016790A, 4α-PDD, or analogs and / or derivatives thereof.

[0105] When a cell population is described herein as having a certain phenotype, it is understood that the phenotype represents a significant portion of the cell population, such as at least 25%, at least 40%, or at least about 50%, or at least about 60%, or at least about 75%, or at least about 80%, or at least about 90% of the cell population. Additionally, at each step, the cell population can be enriched for cells of the desired phenotype and / or depleted of cells of an unwanted phenotype such that the cell population contains at least about 75%, or at least about 80%, or at least about 90% of the desired phenotype. Such positive and negative selection methods are known in the art. For example, fluorescence-activated cell sorting or magnetic beads that bind cells to certain cell surface antigens (including those described herein) can be used based on cell surface antigens to sort cells. A negative selection column can be used to remove cells that express unwanted cell surface markers. In some embodiments, the cells are enriched for CD34+ cells (before and / or after undergoing EHT). In some embodiments, the cell population is cultured under conditions that promote the expansion of CD34+ cells, thereby generating an expanded stem cell population.

[0106] In various embodiments, CD34+ cells (e.g., floating cells and / or adherent cells) are harvested from cultures that undergo the endothelial-to-hematopoietic transition between day 10 and day 20 of iPSC differentiation, such as between day 12 and day 17 of iPSC differentiation.

[0107] In various embodiments, the HSCs or CD34-enriched cells are further expanded. For example, the HSCs or CD34-enriched cells can be expanded according to the methods disclosed in US 8,168,428; US 9,028,811; US10,272,110; and US10,278,990, which are hereby incorporated by reference in their entireties. In some embodiments, the in vitro expansion of HSCs or CD34-enriched cells uses prostaglandin E2 (PGE2) or a PGE2 derivative. In some embodiments of the present disclosure, the HSCs contain at least about 0.01% LT-HSCs, or at least about 0.05% LT-HSCs, or at least about 0.1% LT-HSCs, or at least about 0.5% LT-HSCs, or at least about 1% LT-HSCs.

[0108] Hematopoietic stem cells (HSCs) that give rise to innate myeloid, erythroid, and lymphoid lineages can be identified based on the expression of CD34 and the lack of lineage-specific markers (referred to as Lin-). In some embodiments, the stem cell population containing HSCs is enriched, for example, as described in US 9,834,754, which is hereby incorporated by reference in its entirety. For example, the method can include sorting the cell population based on the expression of one or more of CD34, CD90, CD38, and CD43. Cells that are CD34 + and CD90+ , CD38 - and CD43 - One or more components of are used for further differentiation. In some embodiments, the stem cell population for differentiation into hematopoietic lineages is at least about 80% CD34 - , or at least about 90% CD34 + , or at least about 95% CD34 + .

[0109] In some embodiments, the stem cell population, or CD34-enriched cells or fractions thereof, or derived cell populations are expanded as described in US2020 / 0308540, which is hereby incorporated by reference in its entirety. For example, cells are expanded by exposing the cells to an aryl hydrocarbon receptor antagonist including, for example, SR1 or an SR1 derivative. See also Wagner et al., Cell Stem Cell 2016;18(1):144-55 and Boitano A., et al., Aryl Hydrocarbon Receptor Antagonists Promote the Expansionof Human Hematopoietic Stem Cells .Science September 10, 2010;329(5997):1345–1348.

[0110] In some embodiments, compounds that promote CD34 + cell expansion include pyrimidoindole derivatives, including, for example, UM171 or UM729 (see US2020 / 0308540, which is hereby incorporated by reference).

[0111] In some embodiments, the stem cell population or CD34+-enriched cells are further enriched for cells expressing periostin and / or platelet-derived growth factor receptor alpha (pdgfra), or are modified to express periostin and / or pdgfra, as described in WO 2020 / 205969 (which is hereby incorporated by reference in its entirety). Such expression can be carried out by delivering the coding transcript to the cell, or by introducing a coding transgene, or by a transgene-free method (not limited to introducing non-integrating episomes into the cell). In some embodiments, gene editing is used to introduce genetic modifications into the expression elements in the cell, such as to modify promoter activity or strength, ribosome binding, RNA stability, or to affect RNA splicing.

[0112] In other embodiments, the stem cell population or CD34-enriched cells are cultured with an inhibitor of histone methyltransferase EZH1. Alternatively, EZH1 is partially or completely absent, inactivated, or transiently silenced in the stem cell population. Inhibition of EZH1 can direct bone marrow progenitors (e.g., CD34+CD45+) to the lymphoid lineage. See WO 2018 / 048828, which is hereby incorporated by reference in its entirety. In other embodiments, EZH1 is overexpressed in the stem cell population.

[0113] In various embodiments, the HSC population or a fraction thereof differentiates into hematopoietic lineages, which can be selected from common myeloid progenitors (CMPs), lymphoid-primed multipotent progenitors (LMPPs), granulocyte macrophage DC progenitors (GMDPs), granulocyte / macrophage lineage-restricted progenitors (GMPs), megakaryocyte / erythroid progenitors (MEPs), macrophage / dendritic cell (DC) progenitors (MDPs), common DC progenitors (CDPs), conventional (or classical) myeloid dendritic cells (cDCs), common monocyte progenitors (cMoPs), and plasmacytoid DCs (pDCs) and fractions thereof, from which monocytes, macrophages, and dendritic cells can be generated.

[0114] Differentiation of iPSCs (e.g., into EBs) can employ a WNT agonist such as CHIR99021. A WNT agonist is a molecule that mimics or increases WNT signaling. Non-limiting examples of WNT agonists include the small molecule CHIR-99021 (CAS 252917-06-9), 2-amino-4,6-disubstituted pyrimidines such as BML 284 (CAS 853220-52-7), SKL2001 (CAS 909089-13-0), WAY 262611 (CAS 1123231-07-1), WAY 316606 (CAS 915759-45-4), SB 216763 (CAS 280744-09-4), IQ 1 (CAS 331001-62-8), QS 11 (CAS 944328-88-5), deoxycholic acid (CAS 83-44-3), BIO (CAS 667463-62-9), kenpaullone (CAS 142273-20-9), or (hetero)aryl pyrimidines. In some embodiments, the WNT agonist is an agonist antibody or a functional fragment thereof or an antibody-like polypeptide.

[0115] Differentiation of iPSCs (e.g., differentiation into EBs) can be carried out using ROCK inhibitors. Exemplary ROCK inhibitors for establishing and differentiating iPSCs include, but are not limited to: thiazovivin, Y27632, fasudil, AR122-86, RevitaCell.TM. Supplement, H-1152, Y-30141, Wf-536, HA-1077, hydroxy-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, H-100, and the ROCK inhibitor disclosed in U.S. Patent No. 8,044,201, which is hereby incorporated by reference in its entirety.

[0116] In some embodiments, common myeloid progenitors (CMPs), lymphoid-primed multipotent progenitors (LMPPs), granulocyte macrophage dendritic cell progenitors (GMDPs), granulocyte / macrophage lineage-restricted progenitors (GMPs), megakaryocyte / erythrocyte progenitors (MEPs), macrophage / dendritic cell (DC) progenitors (MDPs), common DC progenitors (CDPs), conventional (or classical) myeloid dendritic cells (cDCs), common monocyte progenitors (cMoPs), or plasmacytoid DCs (pDCs) are cultured in vitro with Notch ligands (in part or in whole), SHH, extracellular matrix components, and / or combinations thereof to effect cell differentiation. Additionally, feeder cells of xenogeneic OP9-DL1 or STO murine fibroblasts or blood-derived peripheral blood mononuclear cells (PBMCs), or mesenchymal stem cells derived from umbilical cord blood or lymphocyte-derived cancer cell line cells are typically used to differentiate hematopoietic cells into innate myeloid cells, T cells, or NK cells, and can optionally be used to differentiate cells into other lineages. The OP9-DL1 co-culture system uses a bone marrow stromal cell line (OP9) transduced with the Notch ligand delta-like 1 (DLL1) to support T cell development from stem cell sources. The OP9-DL1 system limits the potential for clinical application of cells. There is a need for a feeder-free cell system that can generate phagocytic cells (e.g., monocytes, macrophages, dendritic cells, and neutrophils) from hiPSCs for clinical use, and in some embodiments, the present invention meets this objective. In a non-limiting example, to generate mature phagocytic cells using Notch ligands, iPSCs are expanded for 6 days, followed by embryoid body formation, which takes approximately 8 days. The cells are further cultured for approximately 5 days to enable HSCs to develop from CD34+ hematopoietic endothelial cells from which they are derived. The HSCs are then cultured in a specific medium (which can contain Notch ligands) to differentiate into myeloid cells such as monocytes, macrophages, dendritic cells, and neutrophils (as described above).

[0117] In some embodiments, the presence of cytokines and / or growth factors is required, and these include but are not limited to stem cell factor, Fms-like tyrosine kinase 3 ligand, VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15; and optionally, a BMP activator, to initiate the differentiation of permanent hematopoietic endothelium into common myeloid progenitor (CMP), lymphoid-primed multipotent progenitor (LMPP), granulocyte macrophage DC progenitor (GMDP), granulocyte / macrophage lineage-restricted progenitor (GMP), megakaryocyte / erythrocyte progenitor (MEP), macrophage / dendritic cell (DC) progenitor (MDP), common DC progenitor (CDP), conventional (or classical) myeloid dendritic cell (cDC), common monocyte progenitor (cMoP), or plasmacytoid DC (pDC). In some embodiments, a pluripotent stem cell-derived phagocyte progenitor or phagocyte precursor is contacted with a composition comprising one or more growth factors and cytokines (selected from SCF, Flt3L, IL3, IL7, and IL15), wherein the culture medium is free of one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor, to initiate the differentiation of the phagocyte progenitor or phagocyte precursor into monocytes, macrophages, or dendritic cells.

[0118] As used herein, the term "Notch ligand" refers to a ligand capable of binding to a Notch receptor polypeptide present in the T cell membrane of a hematopoietic stem cell or progenitor cell. Notch receptors include Notch-1, Notch-2, Notch-3, and Notch-4. Notch ligands generally have a DSL domain (D - delta, S - Serrate, and L - Lag2), which contains 20 to 22 amino acids at the amino terminus and 3 to 8 EGF repeats on the extracellular surface. In various embodiments, the Notch ligand comprises at least one of delta-like-1 (DLL1), delta-like-4 (DLL4), delta Max (disclosed in PCT / US2020 / 041765 and PCT / US2020 / 030977, which are incorporated herein by reference in their entirety) or a functional portion thereof, Jagged 1 (JAG1), Jagged 2 (JAG2), delta-like ligand 3 (DLL3), and X - delta2. The key signal transmitted by thymic stromal cells to incoming lymphocyte progenitors in vivo is mediated by DL4, which is expressed by cortical thymic epithelial cells.

[0119] As used herein, "Notch ligand" also includes full-length (full-length), partial (truncated form), or modified (including one or more mutations, such as conservative mutations) Notch ligands, as well as Notch ligands of any species or fragments thereof that retain at least one activity or function of the full-length Notch ligand. Also included are peptides that mimic Notch ligands. Notch ligands can be "canonical Notch ligands" or "non-canonical Notch ligands". Canonical Notch ligands are characterized by an extracellular domain that typically includes an N-terminal (NT) domain, followed by a delta / serrate / LAG-2 (DSL) domain and multiple tandemly arranged epidermal growth factor (EGF)-like repeats. Binding of a canonical ligand to Notch generally requires the DSL domain and flanking NT domain, as well as the first two EGF repeats containing the delta and OSM-11-like protein (DOS) motif. The intracellular domain of some canonical ligands contains a carboxyl-terminal PSD-95 / Dlg / ZO-1-ligand (PDZL) motif, the function of which is independent of Notch signaling.

[0120] In some embodiments, the Notch ligand is an anti-Notch (agonist) antibody that can bind to and participate in Notch signaling. In some embodiments, the antibody is a monoclonal antibody (including human or humanized antibodies), a single-chain antibody (scFv), a nanobody, or other antibody fragment or antigen-binding molecule capable of activating the Notch signaling pathway.

[0121] In some embodiments, the Notch ligand is a delta family Notch ligand. In some embodiments, the delta family ligands are delta-1 (Genbank accession number AF003522, Homo sapiens), delta-like 1 (DLL1, Genbank accession numbers NM_005618 and NP_005609, Homo sapiens; Genbank accession numbers X80903, 148324, Mus musculus), delta-4 (Genbank accession numbers AF273454, BAB18580, Mus musculus; Genbank accession numbers AF279305, AAF81912, Homo sapiens) and / or delta-like 4 (DLL4; Genbank accession numbers Q9NR61, AAF76427, AF253468, NM_019074, Homo sapiens; Genbank accession number NM 019454, Mus musculus). Notch ligands are commercially available or can be produced, for example, by recombinant DNA technology.

[0122] In some embodiments, the Notch ligand comprises an amino acid sequence that is at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97% identical (e.g., about 100% identical) to the human DLL1 or DLL4 Notch ligand. Functional derivatives of the Notch ligand (including fragments or portions thereof) will be capable of binding and activating the Notch receptor. Binding to the Notch receptor can be determined by a variety of methods known in the art, including in vitro binding assays and receptor activation / cell signaling assays.

[0123] In various embodiments, the Notch ligand is soluble and optionally immobilized on microparticles or nanoparticles, which are optionally paramagnetic to allow for magnetic enrichment or concentration processes. In other embodiments, the Notch ligand is immobilized on a 2D or 3D culture surface, optionally together with other adhesion molecules such as VCAM-1. See US2020 / 0399599, which is hereby incorporated by reference in its entirety. In other embodiments, the beads or particles are polymers (e.g., polystyrene or PLGA), gold, dextran iron, or are composed of biomaterials such as particles formed from lipids and / or proteins. In various embodiments, the particles have a diameter or maximum dimension ranging from about 0.01 μm (10 nm) to about 500 μm (e.g., about 1 μm to about 7 μm). In other embodiments, a polymeric scaffold with conjugated ligands can be employed as described in WO 2020 / 131582, which is hereby incorporated by reference in its entirety. For example, the scaffold can be composed of polylactic acid, polyglycolic acid, PLGA, alginate or alginate derivatives, gelatin, collagen, agarose, hyaluronic acid, poly(lysine), polyhydroxybutyrate, poly-ε-caprolactone, polyphosphazene, poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide), poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene), Pluronic polyols, poloxamers, poly(uronic acid), poly(anhydride), poly(vinylpyrrolidone), and any combination thereof. In some embodiments, the scaffold includes pores with a diameter between about 1 pm and 100 pm.

[0124] In some embodiments, the C-terminus of the Notch ligand is conjugated to a selected support. In some embodiments, this can include adding a sequence at the C-terminus of the Notch ligand that can be conjugated enzymatically to the support, for example, via a biotin molecule. In another embodiment, a Notch ligand-Fc fusion is prepared such that the Fc fragment can be immobilized by binding to protein A or protein G, which is conjugated to the support. Of course, any known protein conjugation method can be employed.

[0125] In some embodiments, the Notch ligand is DLL4 having one or more affinity-enhancing mutations with respect to hDLL4, such as one or more (or all) of the following: G28S, F107L, I143F, H194Y, L206P, N257P, T271L, F280Y, S301R, and Q305P. See Gonzalez-Perez, et al., Affinity-matured DLL4ligandsas broad-spectrum modulators ofNotch signaling ,Nature Chemical Biology(2022).

[0126] Thus, in various embodiments, the Notch ligand is immobilized, functionalized, and / or embedded in a 2D or 3D culture system. The Notch ligand can be incorporated with components of the extracellular matrix, such as one or more selected from fibronectin, recombinant human fibrin fragment, and laminin. In some embodiments, the Notch ligand and / or components of the extracellular matrix are embedded in an inert material providing 3D culture conditions. Exemplary materials include, but are not limited to, cellulose, alginate, and combinations thereof. In some embodiments, the Notch ligand, components of the extracellular matrix, or a combination thereof are in contact with the culture conditions, providing a topographical pattern and / or texture (e.g., roughness) that aids in the differentiation and / or expansion of cells.

[0127] In various embodiments, the HSC / HSPC population is cultured in an artificial thymic organoid (ATO). See Hagen, M. et al. (2019). The ATO will comprise a culture of HSCs (or HSC aggregates) with a stromal cell line expressing the Notch ligand under serum-free conditions. The artificial thymic organoid is a 3D system that induces the differentiation of hematopoietic precursors into naïve CD3 + CD8 + and CD3 + CD4 + T cells or myeloid lineage cells. In some embodiments, the artificial thymic organoid comprises DLL4 and BMP2, or functional fragments thereof.

[0128] In some aspects and embodiments, the present invention provides a culture platform for obtaining: common myeloid progenitor cells (CMP), lymphoid-primed multipotent progenitor cells (LMPP), granulocyte macrophage dendritic cell progenitor cells (GMDP), granulocyte / macrophage lineage-restricted progenitor cells (GMP), megakaryocyte / erythroid progenitor cells (MEP), macrophage / dendritic cell (DC) progenitor cells (MDP), common DC progenitor cells (CDP), conventional (or classical) myeloid dendritic cells (cDC), common monocyte progenitor cells (cMoP), or plasmacytoid DCs (pDC). The culture platform comprises contacting cells (e.g., CD34+ cells from EBs) with an effective amount of a mechanosensor or a mechanosensitive channel agonist to increase the activity or expression of Dnmt3b. In some embodiments, the mechanosensor is Piezol. Exemplary Piezol agonists include Yoda1, ssRNA40, Jedi1, and Jedi2. In some embodiments, the mechanosensor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. The culture medium may comprise one or more growth factors and cytokines selected from TPO, SCF, Flt3L, IL3, IL-6, IL7, IL-11, IGF, bFGF, and IL15. The culture medium may optionally comprise one or more of VEGF, bFGF, BMP activator, Wnt pathway activator, or ROCK inhibitor (e.g., thiazovivin or Y27632). Subsequently, the cells (e.g., monocytes, macrophages, dendritic cells, and neutrophils or their precursors, as described above) are cultured in the presence of one or more growth factors / cytokines / agonists or inhibitors suitable for differentiation into the desired myeloid cells or their precursors. In some embodiments, the culture medium is suitable for differentiating HSCs into phagocyte progenitor cells (e.g., common myeloid progenitor cells (CMP), lymphoid-primed multipotent progenitor cells (LMPP), granulocyte macrophage dendritic cell progenitor cells (GMDP), granulocyte / macrophage lineage-restricted progenitor cells (GMP), megakaryocyte / erythroid progenitor cells (MEP), macrophage / dendritic cell (DC) progenitor cells (MDP), common DC progenitor cells (CDP), conventional (or classical) myeloid dendritic cells (cDC), common monocyte progenitor cells (cMoP), granulocyte and macrophage progenitor cells (GMP), granulocyte-macrophage progenitor cells (GMP), and plasmacytoid DCs (pDC). For example, at the start of the differentiation culture, IL-3, IL-7, IL-15, SCF, and FLT-3L are added, and in the next stage, the aforementioned cytokines except IL-3 are supplemented to the culture medium.Alternatively, cells are first cultured in SCF, FLt-3L, TPO, GM-CSF, IL-3, and IL-6, and then in one or more of IGF-1, SIS3, IL-7, and IL-21 or granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), interleukin-6 (IL-6), interleukin-4 (IL-4), leukemia inhibitory factor (LIF), and macrophage inflammatory protein-1α (MIP-1α).

[0129] In some embodiments, the HSC population or a fraction thereof differentiates into a myeloid lineage or its progenitor or derivative cells without relying on the use of agonists of mechanosensors or mechanosensitive channels (such as Yoda1). In some embodiments, the use of agonists of mechanosensors or mechanosensitive channels (such as Yoda1) is optional. Thus, in some embodiments, CD34+ cells are enriched from a population of differentiated pluripotent stem cells to prepare a CD34+-enriched population. The CD34+-enriched cell population is induced to undergo an endothelial-to-hematopoietic cell transition for at least two days but not more than 12 days, wherein the use of agonists of mechanosensors or mechanosensitive channels (such as Yoda1, jedi1, jedi2, ssRNA40) is optional. HSCs and / or HSPCs differentiate into a progenitor myeloid lineage cell population or a myeloid lineage cell population.

[0130] In some embodiments, the endothelial-to-hematopoietic cell transition of the CD34+-enriched cell population is induced for at least two days and further for about 4 hours, or about 8 hours, or about 12 hours, or about 16 hours, or about 20 hours, or about 24 hours, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, but in total not more than 12 days. In an exemplary embodiment, EHT is induced for about 4 days to about 10 days, or about 5 days to about 8 days (e.g., in the range of 5 to 7 days).

[0131] In some embodiments, cells express a CAR based on gene editing of iPSCs, embryoid bodies, hCD34+ cells, or myeloid precursors, or via mRNA expression in a target cell population. Additionally, or optionally, cells can be engineered to express cytokines (e.g., IL-4, IL-6, IL-15, etc. or interferons) to make the CAR more effective in targeting tumors.

[0132] In non-limiting examples, cells (such as monocytes, macrophages, dendritic cells, and neutrophils, or their precursors) can be efficiently transduced by vectors such as, but not limited to, retroviral or non-integrating viral vectors (such as adenovirus, adeno-associated virus, integrase-deficient lentivirus, poxvirus) carrying first-generation, second-generation, third-generation, fourth-generation, or fifth-generation CARs, or non-viral vectors (such as plasmid vectors, artificial chromosomes), or episomal or episomal hybrid vectors (see, for example, Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Kostelny et al., Immunol. Rev. 257:127-133 (2014); Sharpe et al., Dis. Model. Meeh. 8(4):337-350 (2015); Brentjens et al., Clin. Cancer Res. 13:5426-5435 (2007); Gade et al., Cancer Res. 65:9080-9088 (2005); Maher et al., Nat. Biotechnol. 20:70-75 (2002); Kostelny et al., J. Immunol. 173:2143-2150 (2004); Sadelain et al., Curr. Opin. Immunol. (2009); Hollyman et al., J. Immunother. 32:169-180 (2009)). Each of these above references is hereby incorporated by reference in its entirety. The CAR can target tumor-associated antigens or markers (such as, for example, CD19, CD38, CD33, CD47, CD20, etc.). According to conventional protocols, CAR expression can be demonstrated in different phagocytic cells (such as, for example, monocytes, macrophages, dendritic cells, and neutrophils) or subsets thereof. CAR cells (such as, for example, CAR.CD19-macrophages, CAR.CD38-macrophages, CAR.CD33-macrophages, CAR.CD47-macrophages, CAR.CD20-macrophages, etc.) may exhibit higher tumor activity (such as, for example, anti-leukemia activity) against CD19, CD38, CD33, CD47, CD20 cell lines and primary blasts obtained from patients (such as those with B-cell precursor ALL) compared to unmodified macrophages.

[0133] The CAR is designed to enhance the ability of cells to recognize, bind to, and kill tumor cells. In some embodiments, the CAR enhances the ability of phagocytic cells (e.g., monocytes, macrophages, dendritic cells, and neutrophils) to recognize tumor cells. In some embodiments, the CAR enhances the anti-tumor activity of phagocytic cells. In some embodiments, but not limited thereto, the CAR is a G protein-coupled receptor 87 (GPR87) CAR, a solute carrier family 7 member 11 (SLC7A11 (xCT)) CAR, a TNF receptor superfamily member 17 (BCMA) CAR, a CD30 CAR, a CD19 CAR, a CD22-CAR, a CD33 CAR, a CD133-CAR, an NKG2D CAR (or a CAR or receptor comprising the NKG2D extracellular domain), a mesothelin-CAR, a CD70 CAR, an NKp30 CAR, a CD73 CAR, or a CAR-phagocytic cell (e.g., a CAR-monocyte, a CAR-macrophage, a CAR-dendritic cell, or a CAR-neutrophil), which targets the following tumors or tumor antigens:

[0134] (i) Human epidermal growth factor receptor 2 (HER2) - ovarian cancer, breast cancer, glioblastoma, colon cancer, osteosarcoma, and medulloblastoma;

[0135] (ii) Epidermal growth factor receptor (EGFR) - non-small cell lung cancer, epithelial cancer, and glioma;

[0136] (iii) Mesothelin - mesothelioma, ovarian cancer, and pancreatic adenocarcinoma;

[0137] (iv) Prostate-specific membrane antigen (PSMA) - prostate cancer;

[0138] (v) Carcinoembryonic antigen (CEA) - pancreatic adenocarcinoma, breast cancer, and colorectal cancer;

[0139] (vi) Glypican-3 - hepatocellular carcinoma;

[0140] (vii) Epidermal growth factor receptor variant III (EGFRvIII) - glioblastoma;

[0141] (viii) Disialoganglioside 2 (GD2) - neuroblastoma and melanoma;

[0142] (ix) Carbonic anhydrase IX (CAIX) - renal cell carcinoma;

[0143] (x) Interleukin-13Ra2 - glioma;

[0144] (xi) Fibroblast activation protein (FAP) - malignant pleural mesothelioma;

[0145] (xii) L1 cell adhesion molecule (L1-CAM) - neuroblastoma, melanoma, and ovarian cancer;

[0146] (xiii) Cancer antigen 125 (CA125) - epithelial ovarian cancer;

[0147] (xiv) Cluster of differentiation 133 (CD133) - glioblastoma, cholangiocarcinoma, adenocarcinoma;

[0148] (xv) Cancer / testis antigen 1B (CTAG1B) - melanoma and ovarian cancer;

[0149] (xvi) Mucin 1 - seminal vesicle cancer;

[0150] (xvii) Folate receptor - a (FR-a) - ovarian cancer;

[0151] (xviii) A growth factor receptor selected from one or more of ErbB1, ErbB2, ErbB3, or ErbB4, IGF1R, IGF2R, TβR I-II, VEGFR1, VEGFR2, VEGFR3, PDGFR (α / β), or FGFR1 to 4. See, for example, Zhou Z et al., Chimeric antigen receptor T cells applied to solid tumors. Front Immunol. October 31, 2022; or Pooria et al., Novel antigens of CAR T cell therapy: New roads; old destination, Translational Oncology, Volume 14, Issue 7, 2021, Zhang C, et al., Chimeric Antigen Receptor T-Cell Therapy. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing, each of which is incorporated herein by reference.

[0152] Thus, in some aspects and embodiments of the present invention, a genetically modified population of phagocytes or their precursors or progeny is engineered to express a chimeric antigen receptor (CAR) on the cell surface, and in particular a CAR that specifically binds to a growth factor receptor. Most typically, the CAR comprises an intracellular domain from the Fcε receptor γ (FcεRIγ). However, in further contemplated embodiments, the CAR may also comprise the intracellular domain of the T cell receptor (TCR) CD3ζ (CD3ζ), alone or in combination with additional components from second- or third-generation CAR constructs (such as CD28, CD134, CD137, and / or ICOS).

[0153] In some embodiments, the CAR comprises at least one domain (e.g., an extracellular domain, a transmembrane domain, and / or an intracellular domain) that inhibits anti-phagocytic signaling in phagocytic cells (e.g., monocytes, macrophages, or dendritic cells). In some embodiments, relative to the same type of cells without the CAR, the CAR improves the effector activity of phagocytic cells (e.g., monocytes, macrophages, or dendritic cells), for example, by inhibiting CD47 and / or SIRPα activity. In some embodiments, the CAR acts as a dominant-negative receptor (e.g., a CD47 sink) by binding to CD47 and inhibiting SIRPα activity.

[0154] In some embodiments, relative to unmodified macrophages, monocytes, or dendritic cells, CAR-modified macrophages, monocytes, dendritic cells, or neutrophils exhibit increased production of one or more inflammatory cytokines. The one or more inflammatory cytokines can be selected from one or more of TNFα, IL-6, IL-1a, IL-1b, IL-12, IL-18, IL-8, IL-2, IL-23, IFNα, IFNβ, IFNγ, IL-2, IL-8, IL33, CCL3, CXCL12, CCL22, CCL4, CXCL10, or CCL2.

[0155] In other aspects, the present invention provides a cell population comprising myeloid cells or their precursors, or a pharmaceutically acceptable composition thereof, which can be produced by the methods described herein. In some embodiments, the cell population is a progenitor myeloid lineage cell population that is capable of engrafting in the thymus, spleen, or secondary lymphoid organs after administration to a subject in need thereof. In various embodiments, a composition for cell therapy is prepared, which comprises the cell population and a pharmaceutically acceptable excipient. The pharmaceutical composition can comprise at least about 10 2 cells, or at least about 10 3 、or at least about 10 4 、or at least about 10 5 、or at least about 10 6 、or at least about 10 7 、or at least about 10 8 cells, or at least about 10 9 cells、or at least about 10 10 cells、or at least about 10 11 cells、or at least about 10 12 cells、or at least about 10 13 cells、or at least about 10 14cells. For example, in some embodiments, a pharmaceutical composition is administered to a subject, and the composition can comprise from about 100,000 to about 400,000 cells per kilogram of subject body weight (e.g., about 200,000 cells / kg). In other embodiments, from about 10 5 to about 5×10 5 cells (e.g., about 2.5×10 5 cells / kg), or from about 10 6 to about 5×10 6 cells (e.g., about 2.55×10 6 cells / kg), or from about 5×10 6 to about 10 7 cells (e.g., about 5×10 6 cells / kg), or from about 10 7 to about 10 8 cells (e.g., about 5×10 7 cells / kg), or from about 10 8 to about 10 9 cells (e.g., about 5×10 8 cells / kg), or from about 10 9 to about 10 10 cells, or from about 10 10 to about 10 11 or about 10 11 to about 10 12 cells, or from about 10 12 to about 10 13 cells, or about 10 13 to about 10 14 cells are administered per kilogram of recipient body weight.

[0156] In some embodiments, the cell population is HLA-A neg homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg . In some embodiments, the cell population is further homozygous for HLA-DRB1. In various embodiments, the composition comprises one or more of the myeloid lineages selected from: monocytes, macrophages, dendritic cells, neutrophils, myeloid progenitor cells (CMP), promyelocytes, granulocyte / macrophage lineage-restricted progenitor cells (GMP), macrophage / dendritic cell (DC) progenitor cells (MDP), common DC progenitor cells (CDP), conventional (or classical) myeloid dendritic cells (cDC), common monocyte progenitor cells (cMoP), and plasmacytoid DC (pDC).

[0157] The pharmaceutical composition for the disclosed method may also contain other therapeutic agents for treating a specific target disease. For example, the pharmaceutical composition may also contain cytokines and growth factors (interleukins, interferons, FGF, VEGF, PDGF, PIGF, STAT, etc.). Such additional factors and / or agents may be included in the pharmaceutical composition to produce the advantages of the treatment methods disclosed herein, namely, to provide improved therapeutic effects and reduced systemic toxicity.

[0158] Phagocytes or CAR-phagocytes (e.g., CAR-monocytes, CAR-macrophages, CAR-dendritic cells or CAR-neutrophils) can be formulated, administered, and dispensed in a manner consistent with good medical practice. Factors considered in this case include the specific disease or disorder being treated, the specific mammal being treated (e.g., a human), the clinical condition of the individual patient, the cause of the disease or disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to the physician. The therapeutically effective amount of the cells to be administered will be governed by such considerations.

[0159] In other aspects, the present invention provides a method of cell therapy comprising administering to a human subject in need thereof the cell population or a pharmaceutically acceptable composition thereof as described herein. In various embodiments, the methods described herein are used to treat blood (malignant and non-malignant), bone marrow, immune, and infectious diseases. In various embodiments, the human subject has a condition comprising one or more of lymphopenia, cancer, immunodeficiency, and autoimmune diseases. Examples of diseases include various autoimmune diseases including, but not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), certain forms of juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, certain forms of myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus, discoid lupus erythematosus, certain forms of thyroiditis, certain forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener's). Hematological malignancies that can be treated include, but are not limited to, acute and chronic leukemia, lymphoma, multiple myeloma, and myelodysplastic syndromes. Infectious diseases that can be treated include, but are not limited to, HIV (human immunodeficiency virus), RSV (respiratory syncytial virus), EBV (Epstein-Barr virus), CMV (cytomegalovirus), adenovirus, and BK polyomavirus-related disorders. Other conditions include: skeletal dysplasia, hemoglobinopathies; anemia, including but not limited to iron-deficiency anemia, pernicious anemia, aplastic anemia, sickle cell anemia, vitamin-deficiency anemia, and hemolytic anemia; bone marrow failure syndromes, and certain genetic disorders (e.g., genetic disorders affecting the immune system). In some embodiments, the subject has cancer, such as a hematological malignancy, including but not limited to leukemia, lymphoma, and multiple myeloma; or a solid tumor, including but not limited to brain tumor, prostate tumor, breast tumor, lung tumor, colon tumor, uterine tumor, skin tumor, liver tumor, bone tumor, pancreatic tumor, ovarian tumor, testicular tumor, bladder tumor, kidney tumor, head tumor, neck tumor, stomach tumor, cervical tumor, rectal tumor, laryngeal tumor, or esophageal tumor.

[0160] In some embodiments, the subject has a disorder selected from the following: acute myeloid leukemia; acute lymphoblastic leukemia; chronic myeloid leukemia; chronic lymphocytic leukemia; myeloproliferative disorders; myelodysplastic syndromes; multiple myeloma; non-Hodgkin lymphoma; Hodgkin disease; aplastic anemia; pure red cell aplasia; paroxysmal nocturnal hemoglobinuria; Fanconi anemia; thalassemia major; sickle cell anemia; severe combined immunodeficiency (SCID); Wiskott-Aldrich syndrome; hemophagocytic lymphohistiocytosis; congenital metabolic defects; severe congenital neutropenia; Shwachman-Diamond syndrome; Diamond-Blackfan anemia; and leukocyte adhesion deficiency.

[0161] In embodiments employing HLA-edited cells as described herein, the composition can be matched to the subject at one or more of the remaining HLA loci. For example, in some embodiments, the cells are matched to the HLA-B, HLA-C, and HLA-DRB1 haplotypes.

[0162] The cell compositions of the present disclosure (e.g., prepared according to the present disclosure) can further comprise a pharmaceutically acceptable excipient or carrier. Such excipient or carrier solutions can also contain buffers, diluents, and other suitable additives. A buffer is a solution or liquid whose chemical constitution neutralizes an acid or a base without a significant change in pH. Examples of buffers contemplated by the present invention include, but are not limited to, normal / saline (0.9% NaCl), 5% dextrose in water (D5W), Dulbecco's phosphate buffered saline (PBS), Ringer's solution. The composition can comprise excipients suitable for intravenous infusion or other routes of administration, and the composition can comprise a suitable cryoprotectant. An exemplary carrier is DMSO (e.g., about 10% DMSO). Other carriers can include dimethoxyethane (DME), N,N-dimethylformamide (DMF), or dimethylacetamide, including mixtures or combinations thereof. The cell compositions can be provided in a suitable volume in an implantable device (e.g., a scaffold), or in a bag, or in a vial, test tube, or container, and stored frozen until use.

[0163] Other compounds can be administered together with the cells, such as cytotoxic agents, immunosuppressive agents, and / or cytokines or growth factors (e.g., stem cell factor, thrombopoietin, transforming growth factor (TGF)-α or β, fibroblast growth factor (FGF), angiopoietin (Ang) family growth factors, insulin-like growth factor, granulocyte-macrophage colony-stimulating factor, TNF-α or TNF-β, VEGF, interleukins (e.g., IL-2, 6, 7, 8, 10, 12, 15, etc.) and interferons (e.g., INF-α or INF-γ)). The combination administration includes co-administration using separate formulations or a single pharmaceutical formulation and sequential administration in either order, wherein preferably there is a period of time during which two (or all) of the active agents exert their biological activities simultaneously.

[0164] Agents that can be co-administered with the phagocytes of the present invention (such as hormones, growth factors, and cytokine antibodies) include molecules such as (renin); growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; α-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors, such as factor VIIIc, factor IX, tissue factor (TF), and von Willebrand factor; anticoagulation factors, such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators, such as urokinase or human urinary or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-α and tumor necrosis factor-β; enkephalinase; RANTES (regulated upon activation, normal T cell expressed and secreted); human macrophage inflammatory protein (MIP-1-α); serum albumin, such as human serum albumin; Mullerian duct inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; murine gonadotropin-related peptide; microbial proteins, such as β-lactamase; deoxyribonuclease; IgE; cytotoxic T lymphocyte-associated antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); hormone or growth factor receptors; protein A or D; rheumatoid factor; neurotrophic factors, such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, neurotrophin-5, or neurotrophin-6 (NT-3, NT4, NT-5, or NT-6) or nerve growth factor, such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factors, such as aFGF and bFGF; fibroblast growth factor receptor 2 (FGFR2), epidermal growth factor (EGF); transforming growth factor (TGF), such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; bone morphogenetic protein (BMP), including BMP1, BMP6, BMP7, and BMP receptor 2; insulin-like growth factor-I and insulin-like growth factor-II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein, hepatocyte growth factor (HGF), EpCAM, GD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, CEA, TENB2, EphA receptor, EphB receptor, folate receptor, FOLR1, mesothelin, cripto, alphavbeta6, integrin, VEGF, VEGFR, EGFR, transferrin receptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5;CD proteins, such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80. CD81, CD103, CD105, CD134, CD137, CD138, CD152, TNF-α, IFN-α, GM-CSF, IL-3 or an antibody that binds to one or more tumor-associated antigens or cell surface receptors; erythropoietin; osteogenic factor; immunotoxin; bone morphogenetic protein (BMP); interferons, such as interferon-α, interferon-β and interferon-γ; colony-stimulating factors (CSF), such as M-CSF, GM-CSF and G-CSF; interleukins (IL), such as IL-2, IL-6, IL-12, IL-23, IL-12 / 23p40, IL-17, IL-15, IL-21, IL-1a, IL-1b, IL-18, IL-8, IL-4, IL-3 and IL-5; superoxide dismutase; T cell receptor; surface membrane protein; decay-accelerating factor; viral antigen, such as a part of the HIV envelope; transporter protein; homing receptor; addressin; regulatory protein; integrins, such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; tumor-associated antigens, such as HER2, HER3 or HER4 receptors; endoglin, c-Met, c-kit, 1GF1R, PSGR, NGEP, PSMA, PSCA, LGR5, B7H4, TAG72 (tumor-associated glycoprotein 72) and fragments of any of the polypeptides listed above.;

[0165] Examples of antibodies or fragments thereof that can be administered include, but are not limited to, anti-PD-L1 antibodies, abciximab (Reopro), adalimumab (Humira, Amjevita), alefacept (Amevive), alemtuzumab (Campath), basiliximab (Simulect), belimumab (Benlysta), bezlotoxumab (Zinplava), canakinumab (Ilaris), certolizumab (Cimzia), cetuximab (Erbitux), daclizumab (Zenapax, Zinbryta), denosumab (Prolia, Xgeva), efalizumab (Raptiva), golimumab (Simponi, Simponi Aria), infliximab (Remicade), ipilimumab (Yervoy), ixekizumab (Taltz), natalizumab (Tysabri), nivolumab (Opdivo), olaratumab (Lartruvo), omalizumab (Xolair), palivizumab (Synagis), panitumumab (Vectibix), pembrolizumab (Keytruda), rituximab (Rituxan), tocilizumab (Actemra), trastuzumab (Herceptin), secukinumab (Cosentyx), ranibizumab, abciximab, reslizumab, caplacizumab, infliximab, bevacizumab, dabigatran, idarucizumab, or ustekinumab (Stelara) or combinations thereof.In addition, the antibody can be selected from anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti-EGFR antibody, anti-cathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti-CEA antibody, anti-retinoblastoma protein antibody, anti-ras oncoprotein antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9 / p24 antibody, anti-CD1 antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD41 antibody, anti-LCA / CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD71 antibody, anti-CD95 / Fas antibody, anti-CD99 antibody, anti-CD100 antibody, anti-S-100 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-light chain antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, and anti-Tn antigen antibody.

[0166] If the administration times of the therapeutic agents are such that the pharmacological activities of the additional therapeutic agents and the active ingredients in the pharmaceutical composition overlap in time, thereby exerting a combined therapeutic effect, co-administration does not require simultaneous administration of the therapeutic agents. Generally, each agent will be administered at the dosage determined for that agent and according to a schedule.

[0167] In certain embodiments, prior to CAR therapy, the patient undergoes lymphodepleting chemotherapy (or other known methods) with chemotherapeutic agents such as fludarabine or cyclophosphamide.

[0168] As used herein, the term "about" means ±10% of the relevant value.

[0169] Certain aspects and embodiments of the present disclosure are further described with reference to the following examples.

[0170] Examples

[0171] Example 1 - ETV2 overexpression increases the yield of hematopoietic endothelial cells and enhances the formation of CD34+ cells during iPSC differentiation, but does not affect pluripotency.

[0172] 方法

[0173] iPSCs are developed from hCD34+ cells by addition-based reprogramming known in the art and substantially as described by Yu, et al. Inducedpluripotentstem celllinesderived from human somaticcells , Science 318, 1917-1920, (2007); and J. Yu, et al. Human induced pluripotent stem cells free of vector and transgene sequences . Science 324, 797-801, (2009). Embryoid body and hematopoietic endothelial differentiation are substantially as described in the following literature: R. Sugimura, et al., Haematopoietic stem and progenitor cells from human pluripotent stem cells . Nature 545, 432-438, (2017); C. M. Sturgeon, et al., Wntsignaling controls the specification of definitive andprimitivehematopoiesisfrom human pluripotent stem cells . Nat Biotechnol 32, 554-561, (2014); J. Yu, et al. Induced pluripotent stem cell lines derived from human somatic cells . Science 318, 1917-1920, (2007); and J. Yu, et al. Human induced pluripotent stem cells free ofvector and transgene sequences . Science 324, 797-801, (2009).

[0174] Briefly, hiPSCs are dissociated and resuspended in a medium supplemented with L-glutamine, penicillin / streptomycin, ascorbic acid, human holo-transferrin, monothioglycerol, BMP4, and Y-27632. Next, the cells are seeded in a 10 cm culture dish (EZSPHERE or low attachment plate) for EB formation. On day 1, bFGF and BMP4 are added to the medium. On day 2, the medium is replaced with a medium containing SB431542, CHIR99021, bFGF, and BMP4. On day 4, the cell medium is replaced with a medium supplemented with VEGF and bFGF. On day 6, the cell medium is replaced with a medium supplemented with bFGF, VEGF, interleukin (IL)-6, IGF-1, IL-11, SCF, and EPO. The cells are maintained in an incubator with 5% CO2, 5% O2, and 95% humidity. To harvest CD34+ cells, EBs are dissociated on day 8, the cells are filtered through a 70 μm filter, and CD34+ cells are isolated by CD34 magnetic bead staining.

[0175] result

[0176] Transduce induced pluripotent stem cells (iPSCs) with an adenoviral vector containing ETV2 and GFP sequences under the control of the EF1A promoter. After transduction, approximately 45% of the iPSC cultures were observed to be GFP-positive, confirming ETV2 overexpression (ETV2-OE). Further observations showed that ETV2-OE in iPSC cells retained the pluripotency characteristics of iPSCs, as demonstrated by the expression of the stem cell marker TRA-1-60 ( Figure 1 ). Figure 1 Show FACS plots representing the transduction efficiency of iPSCs with an adenoviral vector overexpressing ETV2 and GFP sequences.

[0177] Next, ETV2-OE-iPSCs (and control iPSCs transduced with a vector carrying the GFP sequence but not ETV2) were differentiated into embryoid bodies and subsequently into hematopoietic endothelial cells (Strugeon et al., 2014). The results showed that overexpression of ETV2 promoted the formation of hematopoietic endothelial cells, as demonstrated by the expression of CD235a - within the CD34 + and CD31 + markers ( Figure 2 ). Specifically, Figure 2 Show representative flow cytometry analysis of hematopoietic endothelial cells (defined here as CD235a-CD34+CD31+), and relative quantification showed that ETV2-OE enhanced the formation of hematopoietic endothelial cells compared to the control.

[0178] In addition, the results showed that ETV2-OE enhanced the formation of CD34 + cells ( Figure 3 ). Figure 3 Show representative flow cytometry analysis of CD34+ cells, and relative quantification showed that ETV2-OE enhanced the formation of CD34+ cells.

[0179] Overall, these data indicate that overexpression of ETV2 in iPSCs does not affect their pluripotency and promotes their ability to undergo hematopoietic endothelial and hematopoietic differentiation.

[0180] Example 2 - iPSC-derived HSCs generated by Piezo1 activation undergo lineage differentiation similar to bone marrow-derived HSCs.

[0181] method

[0182] To analyze EHT, CD34+ cells derived from EB were suspended in a medium containing Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3. After the cells adhered to the bottom of the wells for approximately 4 to 18 hours (by visual inspection), Yoda1 was added to the cultures. After 4 to 7 days, the cells were collected for analysis.

[0183] iPSCs were differentiated into embryoid bodies for 8 days. On day 8, CD34+ cells were harvested from iPSC-derived embryoid bodies and cultured for an additional 5 to 7 days to induce the endothelial-to-hematopoietic cell (EHT) transition. Then, CD34+ cells were harvested from the EHT cultures between days 5 and 7 for further hematopoietic lineage differentiation.

[0184] result

[0185] Figure 4A and Figure 4B showed that iPSC-derived HSCs activated with Piezo1 underwent pro-T cell differentiation similar to bone marrow (BM)-HSCs. In addition, Figure 5A and Figure 5B showed that iPSC-derived HSCs generated by Piezo1 activation underwent T cell differentiation and could be activated with CD3 / CD28 beads similar to BM-HSCs. Figure 6 showed that iPSC-derived HSCs generated by Piezo1 activation could differentiate into functional T cells, as demonstrated by INFγ expression after stimulation with CD3 / CD28 beads. Collectively, these results indicate that Piezo1 activation during HSC formation enhances the ability of HSCs to further differentiate into hematopoietic lineages in vitro.

[0186] Figure 7 A and Figure 7 B showed that HSCs derived from differentiated iPSCs (D8+7 iPSC-CD34+ cells, + or -Yoda 1 or "Y") could differentiate into neutrophils, as identified by the presence of CD15+ and CD11b (neutrophil markers) Figure 7 A) or the release of myeloperoxidase (MPO) by neutrophils Figure 7 B). D8+7 iPSC CD34+ cells were superior to D8-iPSC-CD34+ cells in differentiating into neutrophils.

[0187] Figure 8 A and Figure 8Panel B shows that neutrophils differentiated from HSCs derived from differentiated iPSCs (D8+7iPSC-CD34, + or –Yoda 1) have phagocytic activity similar to that of neutrophils differentiated from bone marrow CD34+ cells.

[0188] Example 3 – Assessment of off-target editing in HLA-edited HSCs

[0189] HLA typing of triply knocked-out (HLA-edited) HSC clones was performed to check for unwanted editing and to ensure that no major editing events, such as deletions, occurred in other regions of chromosome 6. Sequencing methods and analyses were performed to evaluate the extent of gRNA off-target activity, and gRNAs representing low risk of affecting non-target HLA genes were selected.

[0190] Sequencing was performed using in situ break labeling in fixed and permeabilized cells by ligating full-length P5 sequencing adapters to ends of prepared DSBs. Genomic DNA was extracted, fragmented, end-prepared, and ligated using chemically modified semi-functional P7 adapters. The resulting DNA library contained a mixture of functional DSB-labeled fragments (P5:P7) and non-functional genomic DNA fragments (P7:P7). Subsequently, the DNA library enriched for DNA-labeled fragments was subjected to DNA sequencing, removing all foreign, non-functional DNA. Since the library preparation did not contain PCR, each sequencing read obtained corresponded to a single labeled DSB end from a cell. This generated DNA break reads, enabling direct detection and quantification of genomic DSBs by sequencing without the need for error correction and enabling the generation of a clear list of off-target mutations.

[0191] The results of the editing strategies for two representative clones relative to wild-type cells are summarized in Table 1 below.

[0192] Table 1: Clonal HSC HLA knockouts.

[0193]

[0194] Table 2 provides non-limiting examples of gRNAs used in the experiments that can be used to knock out the expression of the designated HLA genes.

[0195] Table 2: Exemplary gRNA sequences

[0196]

[0197]

[0198] The results show that the editing strategy successfully and selectively targeted the HLA-A, DPB1, and DQB1 genes without affecting other HLA genes or introducing major deletions elsewhere.

[0199] Phenotypic analysis of HLA-edited clones by FACS and immunofluorescence confirmed these results. As Fig. 9A and Fig. 9B shown, the overall expression of class I HLA molecules in HLA-edited cells tested positive compared to the overall expression of class I HLA molecules in wild-type cells. Specific expression of HLA-A by immunofluorescence confirmed that HLA-A was not expressed in HLA-edited cells, confirming the finding that the gene editing strategy was only successful in deleting the HLA-A gene. Specifically, Fig. 9A showed that HLA-edited cells were positive for class I HLA to the same extent as wild-type (WT) (i.e., non-HLA-edited) cells. This result indicates that although HLA-A is deleted, other class I molecules such as HLA-B and C are expressed and are not affected by the gene editing strategy.

[0200] To confirm HLA-A gene deletion, specific expression of HLA-A was analyzed by immunofluorescence. As Fig. 9B can be seen, HLA-A was not expressed in HLA-edited clones, indicating that the gene editing strategy was only efficient in specifically deleting the HLA-A gene. Such retention of all class I expression and HLA-A deletion will facilitate patient matching while avoiding NK cell-mediated rejection.

[0201] Example 4 - Evaluation of the pluripotency and immunocompatibility of HLA-edited HSCs

[0202] Evaluate the ability of HLA-edited cells to retain pluripotency. As Fig.10 shown, immunofluorescence evaluation of HLA-edited iPSC clones indicated that they maintained trilineage differentiation, with ectodermal differentiation indicated by nestin-488 and PAX6-594 staining, mesodermal differentiation indicated by GATA-488 staining, and endodermal differentiation indicated by CXCR4-488 and FOX2A-594 staining.

[0203] Class I HLA molecules are expressed on the surface of all nucleated cells, and if the class I HLA molecules do not match between the donor and the recipient, the cells can be recognized and killed by CD8+ T cells. In addition, HLA mismatch can lead to cytokine release syndrome (CRS) and graft-versus-host disease (GVHD). Conversely, complete deletion of HLA-I molecules by B2M KO will make the cells targets of NK cell-mediated cytotoxicity. Retaining all class I expression and HLA-A deletion can facilitate patient matching while preventing NK cell-mediated rejection. Therefore, the immunocompatibility of HLA-edited HSCs was tested by co-culture with peripheral blood mononuclear cells (PBMCs) to evaluate whether immune cells would reject grafts of HLA-edited and wild-type HSCs (gHSCs).

[0204] Wild-type (gHSC) and HLA-edited HSCs were co-cultured with PBMCs that were matched for HLA-B and HLA-C markers but mismatched for HLA-A. B2M KO HSCs lacking HLA class I molecule expression and CIITA KO HSCs lacking HLA class II molecule expression were used as controls to compare the extent of cytotoxicity mediated by HLA-null and HLA-mismatched PBMCs, respectively. Fig.11 Results of PBMC-mediated cytotoxicity assays in co-cultures measured by annexin V staining are shown. The results show that the deletion of HLA-A in HLA-edited HSCs protects the cells from PBMC-mediated cytotoxicity, while WT, B2M KO, and CIITA KO are vulnerable to PBMC-mediated cytotoxicity. HSCs co-cultured with sorted CD8+ T cells from the same PBMC donor protect HLA-edited and B2M KO HSCs from CD8+ T cell cytotoxicity. In contrast, HSCs co-cultured with sorted NK cells protect only WT and HLA-edited cells from NK cell-mediated cytotoxicity.

[0205] In summary, the immunocompatibility results show that CD8+ T cells present in the PBMC sample are responsible for killing cells mismatched for HLA molecules (WT and CIITA KO), while NK cells present in the PBMC are responsible for killing HLA-null cells (B2M KO). However, HLA-edited HSCs are protected from CD8+ T cell-mediated cytotoxicity (because the mismatched HLA-A has been knocked out) and from NK cell-mediated cytotoxicity (because the expression of HLA class I molecules is largely retained).

[0206] Example 5 - Evaluation of the in vivo engraftment potential of HLA-edited HSCs

[0207] To evaluate the engraftment potential of HLA-edited HSCs, the in vivo engraftment ability of the cells was assessed by competitive transplantation against WT HSCs. Equal proportions of mCherry HLA-edited HSCs and wild-type HSCs (gHSCs) were mixed and transplanted into mice, where bone marrow (BM) and peripheral blood samples were recovered and evaluated by FACS to compare the relative amounts of each cell type present in the samples. As Fig.12 shown, both HLA-edited HSCs and WT HSCs contributed to roughly equal engraftment in BM and peripheral blood samples. These results confirm that HLA-edited HSCs (prepared according to the present disclosure) are comparable to WT HSCs in terms of their engraftment and reconstitution potential. Thus, the properties of WT (unedited, parental) HSCs are expected to be consistent with those of the HLA-edited HSCs of the present disclosure for generating the T cell lineage.

[0208] Example 6 - Differentiation of HLA-Edited HSCs into CD4+ / CD8+ T Cells

[0209] Antigen-presenting cells (APCs) present antigens to helper CD4+ T cells via HLA-II molecules. Activation of helper CD4+ T cells promotes the generation of antigen-specific CD8+ T cells, which further develop into antigen-specific CTLs. Similarly, class I HLA molecules are expressed on the surface of all nucleated cells and present peptide fragments of intracellular proteins to CD8+ CTLs. After recognizing the HLA-I-peptide complex expressed on the cell surface, CTLs induce cytotoxic killing of target (infected) cells. Therefore, studies were conducted to determine whether the deletion of HLA-A affects class I peptide presentation by edited HSCs. As Fig.13A and 13B shown, immunopeptidome analysis revealed that the deletion of HLA-A does not affect the overall presentation of class I peptides. HLA-A-edited cells showed comparable peptide and protein presentation when compared to wild-type (un-HLA-edited) HSCs. In addition, as Fig.14A and 14B shown, the deletion of HLA-DQB1 and HLA-DPB1 does not affect the overall presentation of class II peptides by macrophages differentiated from HSCs. Collectively, these data indicate that despite the deletion of HLA-A, HLA-DQ, and HLA-DP molecules, these cells (and the lineages derived from them) retain their ability to present a broad spectrum of class I and class II peptides.

[0210] Example 7 - In Vivo Testing of Antigen-Mediated Immune Responses.

[0211] Fig.15 is a schematic diagram of delayed-type hypersensitivity, showing the sensitization and elicitation phases of antigen presentation. Briefly, after antigen injection, the antigen is processed by antigen-presenting cells (APCs) and presented by MHC class II molecules on the surface of the APCs. CD4+ T cells recognize the peptide-MHC on the antigen-presenting cells (APCs). When challenged with antigen, CD4+ helper T cells are activated, and cytokines recruit macrophages and other immune cells, which induces tissue swelling.

[0212] The delayed type hypersensitivity assay was performed on transplanted mice. Specifically, the mice were sensitized by subcutaneous injection of sheep red blood cells as the antigen. If the mice have a functional immune system, APCs process the antigen and present the peptide antigen to CD4+ T cells. Next, the challenge was performed by subcutaneous injection of the same antigen into the left paw of the mice. At this time, the T cells are activated and secrete cytokines, which recruit macrophages and other immune cells at the antigen injection site, thus causing tissue swelling. In this assay, a functional immune system results in swelling of the left paw, as measured with a microcaliper.

[0213] As Fig.16A and 16B can be seen, the control (non-transplanted) mice did not show swelling of the left paw due to their immunodeficiency. In contrast, the mice transplanted with cord blood CD34+ cells showed tissue swelling and the diameter of their left paw doubled. Similar immune system responses were found in the mice transplanted with both WT (non-edited HSC) and HLA-edited HSC.

[0214] Example 8: HLA-edited HSCs differentiate into hematopoietic lineages, namely promonocytes / macrophages

[0215] Experiments were performed to determine whether HLA deletion affects the ability of HSCs to differentiate into different types of immune cells. Using a method essentially as described in Example 2, HLA-edited HSCs differentiated into promonocytes / macrophages. It was determined that HLA-edited HSCs were able to differentiate into monocyte / macrophage lineages comparable to WT (non-HLA-edited) HSCs, as measured by their CD11b+-CD14+ expression ( Fig.17A ). In addition, the CD11b+-CD14+ gated population showed equivalent HLA-I and HLA-II expression ( Fig. 17B ), indicating that HLA-edited HSCs also retained the overall expression of both class I and class II molecules.

[0216] The overall expression of other class II molecules in HLA-DQB1 and HLA-DPB1 supported by edited HSCs was evaluated by assessing the expression of macrophages differentiated from HSCs. The study design is schematically shown in Fig.18A . It was found that the deletion of HLA-DQB1 and HLA-DPB1 did not affect the expression of other HLA class II molecules ( Fig.18B ). For example, HLA-DR had comparable expression in both WT and HLA-edited cells ( Fig.18C ). In Fig.18B and 18C , CIITA-KO was used as a positive control.

[0217] References

[0218] 1. Nianias, A. & Themeli, M. Induced Pluripotent Stem Cell (iPSC)–Derived Lymphocytes for Adoptive Cell Immunotherapy: Recent Advances and Challenges. Curr Hematol Malig Rep 14, 261–268 (2019).

[0219] 2. Brauer, P.M., Singh, J., Xhiku, S. & - Pflücker, J.C. T Cell Genesis: In Vitro Veritas Est? Trends Immunol 37, 889–901 (2016).

[0220] 3. Kennedy, M. et al. T Lymphocyte Potential Marks the Emergence of Definitive Hematopoietic Progenitors in Human Pluripotent Stem Cell Differentiation Cultures. Cell Reports 2, 1722–1735 (2012).

[0221] 4. Sturgeon, C.M., Ditadi, A., Awong, G., Kennedy, M. & Keller, G. Wnt Signaling Controls the Specification of Definitive and Primitive Hematopoiesis From Human Pluripotent Stem Cells. Nat Biotechnol 32, 554–561 (2014).

[0222] 5. Chang, C.-W., Lai, Y.-S., Lamb, L.S. & Townes, T.M. Broad T-Cell Receptor Repertoire in T-Lymphocytes Derived from Human Induced Pluripotent Stem Cells. PLoS One 9, (2014).

[0223] 6. Nishimura, T. et al. Generation of Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotency and Redifferentiation. Cell Stem Cell 12, 114–126 (2013).

[0224] 7. Themeli, M. et al. Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat Biotechnol 31, 928–933 (2013).

[0225] 8. Vizcardo, R. et al. Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8+ T Cells. Cell Stem Cell 12, 31–36 (2013).

[0226] 9. Montel-Hagen, A. et al. Organoid-induced differentiation of conventional T cells from human pluripotent stem cells. Cell Stem Cell 24, 376-389.e8 (2019).

[0227] 10. Guo, R. et al. Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors. Cell Research 30, 21–33 (2020).

[0228] 11. Nagano, S. et al. High Frequency Production of T Cell-Derived iPSC Clones Capable of Generating Potent Cytotoxic T Cells. Molecular Therapy-Methods & Clinical Development 16, 126–135 (2020).

[0229] 12. Iriguchi, S. et al. A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy. Nature Communications 12, 430 (2021).

Claims

1. A method for preparing a population of innate myeloid lineage cells or their progenitor cells, the method comprising: Enriching CD34+ cells from a population of differentiated pluripotent stem cells (PSCs) to prepare a CD34+-enriched population; Inducing the CD34+-enriched cell population to undergo an endothelial-to-hematopoietic cell transition for at least two days but not more than 12 days to prepare a population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem cell progenitors (HSPCs); and Differentiating the population comprising hematopoietic stem cells (HSCs) and / or hematopoietic stem cell progenitors (HSPCs) into a progenitor myeloid cell population or a myeloid cell population.

2. The method according to claim 1, wherein the PSC population is a human iPSC population derived from lymphocytes, umbilical cord blood cells, peripheral blood mononuclear cells, CD34+ cells, or human primary tissues.

3. The method according to claim 2, wherein the iPSC population is derived from CD34+-enriched cells isolated from peripheral blood.

4. The method according to claim 2 or 3, wherein the iPSCs are homozygous for one or more HLA class I and / or class II genes.

5. The method according to claim 4, wherein the iPSCs are homozygous for HLA-DRB1.

6. The method according to claim 4, wherein the iPSCs are homozygous for both HLA-B and HLA-C.

7. The method according to any one of claims 2 to 4, wherein the iPSCs are gene-edited to delete one or more HLA class I genes, delete one or more class II genes, and / or delete one or more genes that govern the expression or presentation ability of HLA or MHC.

8. The method according to claim 7, wherein the iPSCs comprise a deletion of HLA-A.

9. The method according to claim 7 or 8, wherein the iPSCs comprise a deletion of HLA-DPB1 and / or HLA-DQB1.

10. The method according to any one of claims 2 to 9, wherein the iPSC is gene-edited to be homozygous for both HLA-A neg , HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg , and optionally further homozygous for HLA-DRB1.

11. The method according to claim 7, wherein the one or more genes that govern the expression or presentation ability of HLA or MHC are β2-microglobulin and / or CIITA.

12. The method according to any one of claims 1 to 11, wherein, Differentiating iPSCs into embryoid bodies (EBs), dissociating the EBs, and recovering CD34+ cells.

13. The method according to any one of claims 1 to 12, wherein CD34+ enrichment and endothelial-to-hematopoietic cell transition are induced on days 8 to 15 of iPSC differentiation.

14. The method according to any one of claims 1 to 13, wherein the CD34-enriched population is cultured in a medium comprising one or more of Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3.

15. The method according to claim 14, wherein the endothelial-to-hematopoietic cell transition generates an HSC population that comprises one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells, and hematopoietic stem cell progenitors.

16. The method according to any one of claims 12 to 15, wherein the CD34+ cells are harvested from a culture undergoing the endothelial-to-hematopoietic cell transition, including harvesting CD34+ floating cells and / or adherent cells.

17. The method according to claim 15 or 16, wherein the HSC population comprises long-term hematopoietic stem cells (LT-HSC).

18. The method according to any one of claims 1 to 17, wherein the induction of the endothelial-to-hematopoietic cell transition includes increasing the expression or activity of dnmt3b.

19. The method according to claim 18, wherein the induction of the endothelial-to-hematopoietic cell transition includes applying cyclic stretching to the CD34+-enriched cells.

20. The method according to claim 19, wherein the cyclic stretching is 2D, 3D or 4D cyclic stretching.

21. The method according to any one of claims 1 to 13, wherein the induction of the endothelial-to-hematopoietic cell transition includes Piezo1 activation.

22. The method according to claim 21, wherein the Piezo1 activation is carried out by contacting the CD34+-enriched cells or a fraction thereof with one or more Piezo1 agonists, the one or more Piezo1 agonists optionally selected from Yoda1, Jedi1, Jedi2, ssRNA40 or analogs or derivatives thereof.

23. The method according to any one of claims 1 to 22, wherein the induction of the endothelial-to-hematopoietic cell transition includes Trpv4 activation.

24. The method according to claim 23, wherein the Trpv4 activation is carried out by contacting the CD34+-enriched cells with one or more Trpv4 agonists, the one or more Trpv4 agonists optionally selected from GSK1016790A, 4α-PDD or analogs or derivatives thereof.

25. The method according to any one of claims 1 to 24, wherein the myeloid lineage is selected from one or more of neutrophils, monocytes, macrophages, dendritic cells or their myeloid precursors.

26. The method according to claim 25, wherein the HSC population is differentiated into a population comprising promyelocytes, optionally by culturing the HSC population in a medium comprising stem cell factor (SCF) and IL-3 and optionally granulocyte colony-stimulating factor (G-CSF).

27. The method according to claim 26, wherein the cell population comprising promyelocytes is differentiated into a cell population comprising neutrophils, optionally by culturing the cell population comprising promyelocytes in G-CSF.

28. The method according to claim 27, wherein the cell population comprising promyelocytes is differentiated into a cell population comprising monocytes or macrophages, optionally by culturing the cell population comprising promyelocytes in granulocyte colony-stimulating factor (G-CSF) and macrophage colony-stimulating factor (M-CSF).

29. The method according to claim 27, wherein the cell population comprising promyelocytes differentiates into a cell population comprising dendritic cells, optionally by culturing the cell population comprising promyelocytes in granulocyte colony-stimulating factor (G-CSF) and TNF-α and optionally IL-4.

30. The method according to claim 29, wherein mature dendritic cells are prepared by culturing the cell population comprising dendritic cells in a medium comprising GM-CSF, IL-1β, TNF-α, INF-γ and PGE-2.

31. The method according to any one of claims 25 to 30, wherein the myeloid lineage cells express a chimeric antigen receptor (CAR).

32. A composition comprising a cell population, the cell population comprising myeloid lineage cells produced by the method according to any one of claims 1 to 31 and a pharmaceutically acceptable carrier.

33. A composition comprising a myeloid lineage that is HLA-A neg and homozygous for both HLA-B and HLA-C, and HLA-DPB1 neg and HLA-DQB1 neg , and optionally further homozygous for HLA-DRB1.

34. The composition according to claim 33, wherein the myeloid lineage is selected from one or more of monocytes, macrophages, dendritic cells, neutrophils and myeloid progenitor cells.

35. The composition according to claim 34, wherein the myeloid progenitor cells are selected from one or more of the following: (CMP), promyelocytes, granulocyte / macrophage lineage-restricted progenitor cells (GMP), macrophage / dendritic cell (DC) progenitor cells (MDP), common DC progenitor cells (CDP), conventional (or classical) myeloid dendritic cells (cDC), common monocyte progenitor cells (cMoP) and plasmacytoid DC (pDC).

36. A method for cell therapy, the method comprising administering to a human subject in need thereof the composition according to claims 32 to 35.

37. The method according to claim 36, wherein the human subject has a condition comprising one or more of lymphopenia, cancer, immunodeficiency, autoimmune disease, viral infection, skeletal dysplasia and bone marrow failure syndrome.

38. The method according to claim 36, wherein the subject has cancer, which is optionally a hematological malignancy or a solid tumor.

39. The method according to any one of claims 36 to 38, wherein the composition is matched to the subject at one or more loci selected from HLA-B, HLA-C and HLA-DRB1.

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