B cell lineage derived from pluripotent cells

Abstract

The present disclosure provides efficient in vitro methods for generating B 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 or lymphatic organs. 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 / 413,454, 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-017PC_Sequence_Listing.xml and is 30,036 bytes in size. BACKGROUND OF THE INVENTION

[0005] The B cell lineage plays a crucial role in tissue maintenance and helps to coordinate effector and regulatory immune responses. However, their clinical use as cell therapies is hampered by the small numbers of such cells that can be isolated from conventional leukocyte-depleted products. Therefore, the development of large-scale, off-the-shelf B lymphocyte lineages would be an attractive tool for combating cancer and infectious diseases, among others. 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 B cells or their progenitors and precursors thereof. 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 generated by the methods disclosed herein, as well as methods for cell therapy.

[0007] In one aspect, the present disclosure provides a method of 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 for CD34+ cells to thereby prepare a CD34+-enriched population. Endothelial-to-hematopoietic transition (EHT) is induced in the CD34+-enriched population to thereby prepare a population of hematopoietic stem cells (HSCs), and optionally further enriching for CD34+ cells thereafter. The resulting HSC population (or a fraction thereof) can differentiate into myeloid cells of the innate immune system (e.g., phagocytes or their precursors). In some embodiments, the present disclosure provides a method of generating B cells, B-CAR cells, and immature and mature B cells (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, the 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, 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. In some embodiments, the 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, the iPSCs are further homozygous for HLA-DRB1.

[0010] In various embodiments, a culture system is used to prepare and expand the iPSCs. 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 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 on days 7 to 14 of iPSC differentiation. Differentiation of iPSCs can be performed according to known techniques. In some embodiments, iPSC differentiation involves a combination of factors such as, but not limited to, Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3.

[0012] Induction of EHT can be performed by any known method. In some embodiments, induction of EHT produces a population of hematopoietic stem cells (HSCs) that includes LT-HSCs. In some embodiments, EHT produces 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 includes 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 performed 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 cultures that undergo an endothelial-to-hematopoietic cell transition between days 10 to 20 of iPSC differentiation, such as days 12 to 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, in particular, the B cell lineage and its progenitors and progeny, including multipotent progenitors (MPP), common lymphoid progenitors (CLP), common lymphoid 2 progenitors (LCA-2), and B cells. The B cells generated can be early pro-B cells, late pro-B cells, pre-B cells, and immature B cells capable of generating B cells. In various embodiments, the present disclosure provides methods for generating in vitro cell populations corresponding to transitional B cells, regulatory B cells, marginal zone B cells, follicular B cells, activated B cells, memory B cells, and plasma B cells (collectively referred to as "B cells").

[0016] In some embodiments, the B cells are further modified to express a chimeric antigen receptor (CAR). Additionally, or optionally, the B-CAR can be engineered to express and / or secrete cytokines (e.g., IL-4, IL-6, IL-15, etc. or interferons) to make the CAR-expressing cells more effective in targeting tumors, for example. In non-limiting examples, the cells can be effectively transduced by a vector carrying the CAR, such as but not limited to a retroviral or non-integrating viral vector or a non-viral vector. In some embodiments, the CAR can target tumor-associated antigens or markers.

[0017] In other aspects, the present invention provides a cell population or a pharmaceutically acceptable composition thereof comprising the B cell lineage or its precursors, and which can be generated by the methods described herein. In some embodiments, the cell population is capable of implanting into the thymus, spleen, or secondary lymphoid organs upon 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] Other aspects and embodiments of the present disclosure will become apparent from the following detailed disclosure and working examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Showing that overexpression (OE) of ETV2 does not affect pluripotency. Figure 1 FACS plots showing 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 characteristics of iPSCs.

[0021] Figure 2 Showing 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 Showing 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 Showing that iPSC-derived HSCs (activated with Piezo1) from EHT of CD34+ cells undergo pro-T cell differentiation similar to bone marrow (BM)-HSCs. Figure 4A FACS plots of the differentiation efficiency of bone marrow (BM) HSCs and iPSC-HSCs (activated with Piezo1) from EHT of CD34+ cells into CD34+CD7+ pro-T cells. Figure 4B Quantification of CD34+CD7+ cells (%) from (1) BM-HSCs and (2) iPSC-HSCs (EHT of CD34+ cells, activated with Piezo1). Figure 4B Showing the average of three experiments.

[0024] Figure 5A and Figure 5B Showing that iPSC-derived HSCs (activated with Piezo1 in this example) generated from EHT of CD34+ cells undergo T cell differentiation and can be activated by CD3 / CD28 beads similar to BM-HSCs. Figure 5A FACS plots of the activation efficiency (CD3+CD69+ expression) of T cells differentiated from BM-HSCs and iPSC-derived HSCs (generated from EHT of CD34+ cells, activated with Piezo1). Figure 5BQuantification of CD3+CD69+ cells (%) derived from (1) BM-HSC and (2) iPSC-HSC (EHT from D8 CD34+ cells, activated with Piezo1). Figure 5B Shows the mean of three experiments.

[0025] Figure 6 Shows that iPSC-derived HSCs generated by EHT of CD34+ cells (activated with Piezo1 in this example) can differentiate into functional T cells. IFNγ expression is the result of T cell activation after stimulation of the T cell receptor (TCR) via CD3 / CD28 beads. Enhanced expression of IFNγ in T cells differentiated from iPSC-derived HSCs (generated by EHT of CD34+ cells, activated with Piezo1 in this example) enhances the ability to further differentiate into functional lymphocytes. Figure 6 Shows the mean of three experiments.

[0026] Figure 7A and Figure 7B Shows phenotypic analysis of HLA-edited (e.g., triple knockout) cells by FACS and immunofluorescence. Figure 7A 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 degree similar to wild-type cells. Figure 7B Shows cell expression of HLA-A via immunofluorescence, where HLA-A is not expressed in HLA-edited clones.

[0027] Figure 8 Shows that HLA-edited clones retain their pluripotency (maintaining tri-lineage differentiation), as indicated 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.

[0028] Figure 9 Shows 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.

[0029] Figure 10Demonstrate the in vivo transplantation potential of HLA-edited HSCs. Equal proportions of mCherry HLA-edited HSCs and unedited HSCs 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.

[0030] Figure 11A and 11B Show that the deletion of HLA-A does not affect the presentation of class I peptides. Figure 11A Show a schematic diagram of immunopeptidome analysis. Figure 11B Show the results of immunopeptidome analysis, which show minimal differences in the number of peptides and representative proteins presented by class I molecules in WT and HLA-edited cells.

[0031] Figure 12A and 12B Show that the deletion of HLA-DP and DQ does not affect the presentation of class II peptides. Figure 12A Show an immunopeptidome analysis plot. Figure 12B Show 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.

[0032] Figure 13 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.

[0033] Figure 14A and 14B Show that HLA-edited HSCs reconstituted a functional immune system, as confirmed by the DTH response of immunodeficient mice. Figure 14A Show 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 using a microcaliper. As Figure 14A 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 14B Is a graphical evaluation of the Figure 14A shown results.

[0034] Figure 15 Demonstrate the potential of HSCs to differentiate into T cell subtypes. After a 35-day differentiation period, the presence of CD4+, CD8+, and AB+ T cell populations in pro-T cells was evaluated by cell sorting. Figure 15The differentiation potential of bone marrow-derived CD34+ cells, embryoid body CD34+ cells, and HSCs ("gHSCs") prepared according to the present invention (e.g., using Piezo1 activation) was compared.

[0035] Figure 16 The degree of T cell-mediated cytotoxicity measured in co-cultures of T cells derived from HSCs and CD19+ lymphoma cells in the presence of an anti-CD3 / CD-19 bispecific antibody was shown. According to the present disclosure, T cells prepared from hematopoietic HSCs ("gHSCs") exhibited a high level of cytotoxicity against target cells.

[0036] Figure 17 The ability of HSCs to develop into pro-T cells, as measured by their CD34-CD7+ markers, was shown.

[0037] Figure 18A and 18B It was shown that, according to the present disclosure, the expression of T cell-specific transcription factors and thymic seeding molecules was increased in pro-T cells derived from HSCs. Figure 18A TCF7 mRNA expression was shown, Figure 18B CCR7 mRNA expression was shown.

[0038] Figure 19A and 19B It was shown that pro-T cells derived from HSCs seeded and differentiated in the thymus. Figure 19A The seeding and analysis procedures were demonstrated. Figure 19B FACS analysis of the CD3 cell population gated on the CD45+ cell population was shown, which indicated that pro-T cells derived from HSCs had excellent seeding and differentiation potential in the thymus.

[0039] Figure 20 It was shown that T cells derived from HSCs could be activated in vitro. The top inset showed FACS analysis of activated T cells from different sources, including HSCs prepared according to the present disclosure. The T cells of the present disclosure exhibited comparable or superior activation, as measured by increased expression of CD107. The bottom inset showed Dynabeads activation, in which the activated T cells expressed inflammatory cytokines. T cells derived from HSCs expressed higher levels of inflammatory cytokines, as exemplified by the expression levels of TNF-α and interferon γ.

[0040] Figure 21A and 21B It was shown 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 21A ). Figure 21BShow HLA-I and HLA-II analysis of cells gated by CD11b+CD14+.

[0041] Figures 22A to 22C Show that the deletion of HLA-DQB1 and HLA-DPB1 does not affect the expression of other HLA class II molecules. Figure 22A Is a schematic diagram of the differentiation of HLA-edited iPSCs into macrophages. Figure 22B Is an immunofluorescence experiment that confirmed the specific deletion of DPB1 and DQB1 molecules. Figure 22C Show that the same cells retained the expression of class II DRB1.

[0042] Figure 23 Show that HLA-edited HSCs can differentiate into megakaryocytes (MKs), which can further differentiate into platelets. The image on the left shows that, by optical microscopy magnified 1000 times, the proportion of platelets in HSCs increased. The figure on the right shows that, compared with BM CD34+ and iPSC-34+ cell populations, the proportion of platelets differentiated from HLA-edited HSCs was significantly increased statistically.

[0043] Figure 24 Show the ability of HSCs to effectively differentiate into NK cells, as confirmed by fluorescence-activated cell sorting (FACS) experiments (gated based on CD56 expression).

[0044] Figures 25A to 25C Show that HSC-derived naive NK cells effectively kill tumor cells. Figure 25A Show a schematic diagram of an experiment in which HSC-derived NK cells are co-cultured with K562 HLA-null cells, and the degree of NK cell degranulation is measured using annexin V staining and cytotoxicity assays. Figure 25B Show the results of NK cell degranulation, as measured by fluorescence-activated cell sorting (FACS) using annexin V staining. Figure 25C Show the results of tumor cell cytotoxicity assays, where lactate dehydrogenase (LDH) is used as a measure of cell death.

[0045] The term "gHSC" is used herein to refer to the iPSC-derived hematopoietic stem cells of the present disclosure.

[0046] 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.

[0047] EB34+ cells refer to CD34+ cells derived from embryoid bodies. These include hematopoietic endothelial cells.

[0049] The present disclosure provides, in various aspects and embodiments, methods for in vitro generation of hematopoietic lineages for cell therapy, and in particular B cell lineages and their progenitors and progeny, including, in various embodiments, multipotent progenitors (MPP), common lymphoid progenitors (CLP), common lymphoid 2 progenitors (LCA-2), and B cells. The generated B cells can be early pro-B cells, late pre-B cells, pre-B cells, and immature B cells capable of generating B cells. In various embodiments, the present disclosure provides methods for in vitro generation of cell populations corresponding to transitional B cells, regulatory B cells, marginal zone B cells, follicular B cells, activated B cells, memory B cells, and plasma B cells (collectively referred to as "B cells"). In various embodiments, the present invention provides efficient in vitro methods for developing such hematopoietic lineages from human induced pluripotent stem cells (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 lymphoid organs. The present invention also provides isolated cells and cell compositions generated by the methods disclosed herein, as well as methods for cell therapy.

[0050] In accordance with various aspects and embodiments of the present disclosure, the ability of human induced pluripotent stem cells (hiPSCs) to generate substantially unlimited amounts of pluripotent stem cells (PSCs) is used to generate an unlimited supply of B cell lineages or modified versions thereof (e.g., gene-modified B-CAR cells). The use of B cells as therapeutic lymphocytes is limited by their limited availability, cell numbers, restricted expansion potential, and histocompatibility issues. In addition, hiPSCs can be more easily genetically modified in vitro compared to primary cells, thus providing improved cell targeting specificity, cell numbers, and bypassing, for example, HLA matching issues. In addition, compared to primary cells, fully engineered hiPSC clones can serve as a stable and safe source (Nianias and Themeli, 2019). In addition, 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, the use of hiPSCs according to the present disclosure has several advantages over primary cells for generating therapeutic hematopoietic lineages such as B cell lineages.

[0051] In one aspect, the present disclosure provides a method for preparing a cell population of B cell lineage (e.g., in vitro). 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 prepare a CD34+-enriched population. Endothelial-to-hematopoietic cell transition (EHT) is induced in the CD34+-enriched population to 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 a B cell lineage.

[0052] In various embodiments, the B cell population includes transitional B cells, regulatory B cells, marginal zone B cells, follicular B cells, activated B cells, memory B cells or plasma B cells, or derivatives thereof.

[0053] In some embodiments, the cell population includes transitional B cells (TrB cells), which are immature B cells and precursors of mature B cells. TrB cells account for approximately 4% of all CD19+ B lymphocytes in healthy individuals. They are present in peripheral blood, cord blood, bone marrow, and secondary lymphoid tissues such as lymph nodes, spleen, tonsils, and gut-associated lymphoid tissue (GALT). Human TrB cells are typically characterized by the hi CD38 hi CD24 phenotype. Based on the expression of CD27, IgM, IgD, CD10, CD21, and CD32, TrB cells can be divided into various subsets. T1 - T3 B cell subsets express low levels of CD27, while CD27+ TrB cells express CD27, CD24, and CD38 at higher levels. In T1 B cells, IgM, CD10, and CD32 are highly expressed, while IgD and CD21 are expressed at lower levels. In T2 B cells, IgM, IgD, CD10, and CD32 are moderately expressed, and CD21 is expressed at a lower level. T3 B cells express IgM, IgD, CD10, CD21, and CD32 at low levels.

[0054] TrB cells can inhibit autoreactive CD4+ T cell proliferation; inhibit the production of pro-inflammatory cytokines by restricting the expansion of CD4+ Th1 cells (production of IFN-γ and TNF-α) and CD4+ Th17 cells (production of IL-17); prevent the differentiation of CD4+ T cells into Th1 and Th17 cells, and promote the conversion of effector CD4+ T cells into CD4+ FoxP3+ Tregs while restricting the production of excessive pro-inflammatory cytokines. TrB cells also inhibit CD8+ T cell responses and maintain invariant natural killer T (iNKT) cells. In addition to producing anti-inflammatory factors, TrB cells also secrete pro-inflammatory cytokines such as IL-6 and TNF-α. In some aspects and embodiments, TrB cells are closely related to interleukin-10-producing regulatory B cells (Bregs) in terms of phenotypic and functional similarities. TrB cells also produce IL-10 and regulate CD4+ T cell proliferation and differentiation into helper T (Th) effector cells.

[0055] In some embodiments, the cell colony comprises regulatory B (Breg) cells. Breg cells are immunosuppressive cells that support immune tolerance. Breg cells are also associated with the suppression of excessive inflammation. By producing IL-10, TGF-β and IL-35, Breg cells can inhibit the differentiation of proinflammatory lymphocytes, such as monocytes producing tumor necrosis factor α (TNF-α), dendritic cells producing IL-12, Th17 cells, Th1 cells and cytotoxic CD8+T cells. Breg cells can also induce the differentiation of immunosuppressive T cells, Foxp3+T cells and regulatory T cells 1 (Tr1). Breg cells also support the maintenance of iNKT cells. Common markers for human Breg cells include CD19+CD24 hi CD38 hi CD1d hi 、CD19+CD24 hi CD27+、CD24 hi CD27+、CD19+CD24 hi CD27 int 、CD19+CD24 hi CD38 hi and CD19+CD25 hi CD71 hi .

[0056] In various embodiments, the B cells have a phenotype consistent with marginal zone B (MZB) cells. MZB cells provide the first line of defense in response to infections with blood-borne viruses and encapsulated bacteria, where they rapidly produce IgM and class-switched IgG antibodies. MZB cells can also produce IgM and class-switched IgG and IgA antibodies in response to commensal antigens. MZB cells mediate the production of T cell-dependent antibodies. For example, MZB cells can initiate T cell-dependent responses to microbial protein antigens. In some embodiments, MZB-like cells derived from iPSCs are CD27+IgM+IgD+ cells, or they express high levels of IgM, CD21, CD1, and CD9 ,and IgD , CD23 , low or negative expression of CD5 and CD11b, or CD27 - CD45RB + (which defines MZ precursor cells).

[0057] In some embodiments, the B cells have a phenotype consistent with follicular B cells. Follicular B cells are involved in T cell-dependent antibody responses. In addition, follicular B cells respond to blood-borne pathogens in a T cell-independent manner. Upon activation, follicular B cells differentiate into short-lived plasma cells in the periphery or enter the T cell-dependent germinal center reaction. Follicular B cells express high levels of IgD and CD23; lower levels of CD21 and IgM; and do not express CD1 or CD5. Other cell surface markers that can identify follicular B cells include, but are not limited to, CD10, CD19, CD20, CD22, CD23, CD38 LOW , CXCR5+ and IgD high .

[0058] B cells express the B cell receptor (BCR), which activates the B cell upon binding to soluble or membrane-bound antigen. The activated BCR forms microclusters and triggers downstream signaling cascades. The microclusters ultimately undergo a contraction phase and form an immunological synapse, which enables stable interaction between the B cell and the T cell, providing bidirectional activation signals. Upon encountering antigen, mature activated B cells proliferate and become blast B cells. These B cells form germinal centers. Germinal center B cells undergo somatic hypermutation and class switch recombination. Plasma cells and memory B cells with high affinity for the original antigenic stimulus are generated. These cells have a long lifespan, and plasma cells may still secrete antibodies for weeks after the primary infection. One of the major transcriptional activators associated with B cell activation is nuclear factor (NF)-κB. Some common markers that identify activated B cells are CD19, CD25, and CD30.

[0059] The B cells generated according to the present disclosure can differentiate into plasma cells (in vitro or in vivo). Plasma cells are specialized terminally differentiated B cells that synthesize and secrete antibodies to maintain humoral immunity. Plasma B cells absorb antigen by receptor-mediated endocytosis upon encountering a specific antigen. The antigenic particles are transferred to the cell surface, loaded onto MHC II molecules, and presented to helper T cells. The binding of the helper T cell to the MHC II-antigen complex activates the B cell. The activated B cell undergoes a period of rapid proliferation and somatic hypermutation. Cells that produce antibodies with high affinity for the specific antigen are selected. Once terminally differentiated, plasma B cells secrete only antibodies specific for that antigen and can no longer produce antibodies against other antigens.

[0060] B cells generated according to the present disclosure can differentiate into memory B cells (in vitro or in vivo). Memory B cells are B lymphocytes that remember specific antigens after an initial B cell response. Memory B cells are stored in the germinal centers of the lymphatic system for when the immune system encounters the specific antigen again. Memory B cells are marked by displaying and secreting antibodies with significantly higher affinity than those produced by primary plasma cells. During any repeated exposure, follicular helper T cells prompt memory cells to differentiate into plasma B cells that are more sensitive to the specific antigen. This rapidly activates the immune system to initiate a faster and stronger response than ever before. In humans, memory B cells are typically identified by CD27 expression, low-level expression of CD23 / FcεRI, or absence of expression of plasma cell markers (syndecan-1 / CD138). DEP-1 / CD148 is also frequently used to identify human memory B cells, as are high-level expressions of B7-1 / CD80, B7-2 / CD86, and CD95. Different subsets of memory B cells and plasma cells can be identified based on the expression of their Ig subtypes (IgM, IgD, IgG, IgA), which is well understood by those skilled in the art.

[0061] 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 B lymphocytes, umbilical cord blood cells (e.g., CD34+ cells), PBMCs or fractions thereof, 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 assist with 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 knock out other functions 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. In some embodiments, iPSCs are prepared from B cells or other cells encoding a defined BCR or antibody, where the BCR or antibody has a predetermined antigen specificity (e.g., against an infectious disease antigen, such as a bacterial or viral surface protein). The iPSCs prepared from such B cells will give rise to B cells with a defined antigen specificity when differentiated into the B cell lineage.

[0062] 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 remaining 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-.

[0063] In some embodiments, the iPSCs are gene-edited to be HLA-A neg , homozygous for both HLA-B and HLA-C, and gene-edited to be HLA-DPB1 neg and HLA-DQB1 neg . In some embodiments, the iPSCs are further homozygous for HLA-DRB1.

[0064] 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 the cell line or population, and thus the cell line or population does not exhibit significant functional expression of the gene. Such cells can be produced 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 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 critical cis-acting expression control sequences.

[0065] 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 No. 10,676,165; U.S. Patent No. 9,580,689; and U.S. Patent No. 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 that have limited replication ability and thus are lost after several generations of cells.

[0066] In some embodiments, human pluripotent stem cells (e.g., iPSCs) are gene-edited. Gene editing includes, but is not limited to, for example, modification of HLA genes (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 B cell receptor genes, or addition of a chimeric antigen receptor (CAR) gene. Exemplary CAR-B cells can be specific for tissues of an inflammatory or infected tissue or can be specific for a target pathogen or cell. For example, the iPSC can be a B cell receptor-transduced iPSC. Such embodiments enable large-scale production of regenerative B lymphocytes with the desired antigen specificity. Alternatively, engineered iPSCs with one or more HLA knockouts can be placed in a bioreactor and differentiated in feeder-free and serum-free conditions under GMP-grade conditions to produce fully functional B cells (e.g., TrB cells, Bregs, plasma B cells, memory B cells, or B cell progenitors).

[0067] In some embodiments, the iPSCs are gene edited using a gRNA that is 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: a "crispr RNA" (or "crRNA") and a "tracr RNA", and is well known to those skilled in the art.

[0068] 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), etc. Fusion proteins containing one or more of these DNA binding domains and a cleavage domain of the Fokl endonuclease can be used to create double-strand breaks in a desired region of DNA in a cell (see, e.g., 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 (e.g., CRISPR-Cas9) known in the art. See, e.g., 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 that guides the desired gene editing (while limiting or avoiding off-target editing) is known in the art. See, e.g., Mohr SE et al., CRISPR guide RNA 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 to Streptococcus pyogenes Cas9 or Prevotella and Francisella 1 (Cpf1 or Cas12a), although with low primary sequence homology, 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 the Current 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 Editing, 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 the dead Cas (dCas) system (e.g., Cas fusion proteins) to direct DNA modification enzymes to the desired target, using dCas as a guide RNA-directed system. Brezgin S, Dead Cas Systems: Types, Principles, and Applications ,IntJ Mol Sci. December 2019; 20(23): 6041.

[0069] Base editors that can install precise genomic alterations without generating double-stranded DNA breaks can also be used for gene editing in cells (e.g., iPSCs) (e.g., engineering gene therapy vectors). Base editors essentially comprise catalytically disabled nucleases, such as Cas9 nickase (nCas9), which cannot generate DSBs, and are fused to a nucleobase deaminase and, in some cases, to 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. Prime 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, as described in WO 2020 / 191153, are also contemplated.

[0070] 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.

[0071] To improve 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 for directly delivering RNPs to cells. For example, RNPs can be delivered into 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 that genome cleavage can begin immediately. See, e.g., Zhang S, Shen J, Li D, Cheng Y. Strategies in the delivery of Cas9 ribonucleoprotein 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.

[0072] 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 is located 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 the haplotype or polymorphism of the HLA locus does not include four Gs, four Cs, GC repeat sequences, or combinations thereof.

[0073] 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.

[0074] gRNAs can be used to develop cloned iPSCs. On-target editing, off-target editing, and 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 the region of interest, followed by next-generation sequencing (e.g., amplicon sequencing, AMP-seq). The on-target and translocation groups 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 group can enrich any potential off-target regions identified by sequencing and allows selection of iPSC clones with negligible off-target mutations. Collectively, these assays enable screening of iPSC clones to select clones with the desired edits while excluding potential CRISPR / Cas9-related genomic integrity issues.

[0075] 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 are different from the CD34+ starting material, enabling informed selection of iPSC clones that do not generate mutations during reprogramming.

[0076] 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 dataset 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.

[0077] In some embodiments, array comparative genomic hybridization (aCGH) analysis is used to select iPSC clones that do not generate copy number aberrations (CNAs) 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 CNAs by comparing sample DNA and reference DNA.

[0078] 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 involves using highly multiplexed PCR to amplify regions associated with hematopoietic malignancies, followed by next - generation sequencing.

[0079] 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 delivering episomal vectors encoding reprogramming factors. However, although rare, episomal vectors can randomly integrate into the cellular 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.

[0080] In some embodiments, after assessing 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 genes for hematological malignancies, 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 hematological malignancy NGS panel AMP-Seq analysis, and / or droplet digital PCR (ddPCR).

[0081] In various embodiments, iPSCs are prepared and expanded using a culture system. The expanded iPSCs can be used 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 US 2019 / 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. Other methods for expansion or differentiation of EBs, including 3D suspension culture, are described in WO 2020 / 086889, which is hereby incorporated by reference in its entirety.

[0082] According to the present disclosure, CD34+ cells are isolated from pluripotent stem cells (e.g., EBs) and induced to differentiate from endothelial cells to hematopoietic cells to prepare a population of hematopoietic stem cells (HSCs). 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.

[0083] 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 a combination 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 non-integrating episomal plasmid encoding 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 for introducing mRNA can be used, 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.

[0084] After CD34+ enrichment, HSCs are generated from endothelial cells using mechanical, biochemical, pharmacological, and / or genetic stimulation or modification.

[0085] 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 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. 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.

[0086] In non-limiting examples, isolated iPSCs can be cultured under conditions that promote lymphohematopoiesis. In some embodiments, feeder cells such as STO mouse fibroblast feeder layers can be used to expand B cells. In some embodiments, hPSCs are co-cultured with murine bone marrow-derived feeder cells such as OP9, 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 media. The cultures can contain growth factors and cytokines to support the differentiation of embryoid bodies or monolayer systems. Feeder cell co-culture systems can be used to generate pluripotent HSPCs, which can further differentiate into several hematopoietic lineages, including B lymphocytes, monocytes or macrophages, dendritic cells, neutrophils, NK cells, T 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 the 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 of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specificationof lineage-restricted precursors ,Cell Stem Cell. October 3, 2013; 13(4).

[0087] Differentiation of iPSCs (e.g., differentiation into EBs) can employ WNT agonists such as CHIR99021. WNT agonists are molecules that mimic or increase 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.

[0088] Differentiation of iPSCs (e.g., differentiation into EBs) can use ROCK inhibitors. Exemplary ROCK inhibitors for the establishment and differentiation of 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.

[0089] Induction of EHT can be performed by any known method. In some embodiments, 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 some embodiments, the EHT culture contains one or more (e.g., in combination) of Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3.

[0090] 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 performed by mechanical, genetic, biochemical, or pharmacological means. In some embodiments, the method includes increasing the activity or expression of DNA (cytosine-5-)-methyltransferase 3 beta (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, induction of EHT includes increasing the expression or activity of dnmt3b.

[0091] 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 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 of the mechanotransduction channel Piezol .eLife (2015).

[0092] 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 antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation , in British J. of Pharmacology 175(1744 - 1759):2018. Other Piezo1 agonists include Jedi1, Jedi2, single - stranded (ss) RNA (e.g., ssRNA40) and their derivatives and analogs. See Wang Y., et al., A lever-like transduction pathway for long-distance chemical-and mechano-gating of the mechanosensitive Piezo1 channel . Nature Communications(2018)9:1300; Sugisawa et al., RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis , Cell, Volume 182, Issue 3, 2020, Pages 609 - 624, which is hereby incorporated by reference in its entirety. These Piezo1 agonists are commercially available. In various embodiments, the effective amount of the 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.

[0093] 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 to at least EBs generated from iPSCs, CD34 + cells isolated from EBs, and / or combinations thereof, which, according to various embodiments, allows for better generation of B - cell lineage cells compared to other methods for inducing EHT. In a non - limiting example, under culture conditions, EHT CD34 + cells are treated with Yoda 1 for 2 to 7 days, 3 to 7 days, 4 to 7 days, 5 to 7 days, or 6 to 7 days to generate advanced HSCs with a greater potential to generate B - cell lineages or their progenitors.

[0094] 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 cells, or by introducing a transgene encoding Dnmt3b, or by a transgene - free method (not limited to introducing non - integrating episomes into cells). In some embodiments, gene editing is employed to introduce genetic modifications to the Dnmt3b expression element in cells, such as but not limited to increasing promoter strength, ribosome binding, RNA stability, and / or affecting RNA splicing.

[0095] In some embodiments, the method includes increasing the activity or expression of Gimap6 in 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, an mRNA transcript encoding Gimap6 can be introduced into cells, or a transgene-free method can be employed, including but not limited to introducing episomes into cells; or alternatively a transgene encoding Gimap6. In some embodiments, gene editing is used to introduce genetic modifications into the Gimap6 expression element in cells (such as one or more modifications to increase promoter strength, ribosome binding, RNA stability or affect RNA splicing).

[0096] In embodiments of the present disclosure that employ mRNA delivery to cells, known chemical modifications can be used to avoid the innate immune response in cells. For example, synthetic RNA containing only canonical nucleotides can bind pattern recognition receptors and can trigger an effective immune response in 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 protein. See US 9,181,319, which is hereby incorporated by reference, particularly with respect to nucleotide modifications to avoid the innate immune response.

[0097] In some embodiments, the expression of Dnmt3b and / or Gimap6 is increased by introducing a transgene into cells, which can direct the desired overexpression level (with other options of different promoter strengths or expression control elements). A variety of 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 a transgene-free method (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.

[0098] In some embodiments, the method includes applying cyclic 2D, 3D, or 4D stretch to 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 WO2017 / 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., a cyclic strain of about 6%), 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.

[0099] 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.

[0100] 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. Negative selection columns can be used to remove cells that express unwanted cell surface markers. In some embodiments, 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. Additionally, these techniques can be used to enrich or isolate subsets of the B cell lineage.

[0101] In various embodiments, CD34+ cells (e.g., floating cells and / or adherent cells) are harvested from cultures that undergo the endothelial-to-hematopoietic cell transition between day 8 and day 20 of iPSC differentiation, such as between day 10 and day 17 of iPSC differentiation.

[0102] In various embodiments, HSCs or CD34-enriched cells are further expanded. For example, HSCs or CD34-enriched cells can be expanded according to methods disclosed in US 8,168,428; US 9,028,811; US10,272,110; and US10,278,990, which patents 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, 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.

[0103] Hematopoietic stem cells (HSCs) that promote lymphopoiesis or give rise to lymphoid lineages can be identified based on the expression of CD34 and the absence of lineage-specific markers (referred to as Lin-). In some embodiments, stem cell populations containing HSCs are enriched, e.g., as described in US 9,834,754, which is hereby incorporated by reference in its entirety. For example, the method can include sorting a cell population based on the expression of one or more of CD34, CD90, CD38, CD43, CD45, CD19, CD20, CD138, or CD10. Those selected as CD34 + , CD90 + , CD38 - , CD19, CD20, CD138, and / or CD43 - fractions of one or more are further differentiated. 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 + .

[0104] In some embodiments, the stem cell population, or CD34+-enriched cells or their components, 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 them to an aryl hydrocarbon receptor antagonist including, e.g., 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 Expansion of Human Hematopoietic Stem Cells .Science Sep 10, 2010;329(5997):1345–1348.

[0105] In some embodiments, compounds that promote CD34 + cell expansion include pyrimidoindole derivatives including, e.g., UM171 or UM729 (see US 2020 / 0308540, which is hereby incorporated by reference).

[0106] 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 employed 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.

[0107] 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 or 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.

[0108] In various embodiments, the HSC population or a fraction thereof differentiates into the B cell lineage or a progenitor thereof, such as a multipotent progenitor (MPP), a common lymphoid progenitor (CLP), a common lymphoid 2 progenitor (LCA-2), an early pro-B cell, a late pro-B cell, a pre-B cell, an immature B cell lineage, and fractions thereof, whereby mature B cells can be produced. The B cells produced according to this method can have a phenotype consistent with transitional B cells, regulatory B cells, marginal zone B cells, follicular B cells, activated B cells, memory B cells, or plasma B cells, as previously described.

[0109] In some embodiments, the HSC population or a fraction thereof differentiates into B cells or a progenitor or derivative thereof without relying on the use of an agonist of a mechanosensitive receptor or mechanosensitive channel (such as Yoda1). In some embodiments, the use of an agonist of a mechanosensitive receptor or mechanosensitive channel (such as Yoda1) is optional. Thus, in some embodiments, CD34+ cells are enriched from a differentiated pluripotent stem cell population 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 no more than 12 days, wherein the use of an agonist of a mechanosensitive receptor or mechanosensitive channel (such as Yoda1, jedi1, jedi2, ssRNA40) is optional. HSCs and / or HSPCs differentiate into a progenitor B cell population or a B cell population.

[0110] In some embodiments, the endothelial-to-hematopoietic cell transition of the CD34+-enriched cell population is induced for at least two days and optionally further continued for at least about 4 hours, or at least about 8 hours, or at least about 12 hours, or at least about 16 hours, or at least about 20 hours, or at least about 24 hours, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days. Generally, the induction of EHT lasts no more than 12 days. In various embodiments, the induction of EHT lasts from 4 days to about 8 days, or from 5 days to about 7 days.

[0111] In some embodiments, the lymphohematopoietic lineage is cultured in vitro with Notch ligand(s) (partial or complete), SHH, extracellular matrix components, and / or combinations thereof to differentiate HSCs into the lymphohematopoietic lineage. Additionally, according to known methods, feeder layers of xenogeneic OP9-DL1 or STO mouse fibroblasts, blood-derived peripheral blood mononuclear cells (PBMCs), or umbilical cord blood-derived mesenchymal stem cells or lymphocyte-derived cancer cell line cells can be used to differentiate hematopoietic cells into the lymphohematopoietic lineage, T cells, or NK cells and can optionally be used to differentiate HSCs 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. Thus, in some embodiments, the method employs a feeder-free cell system for generating B cell lineages for clinical use from hiPSCs. In a non-limiting example, to generate cells of lymphohematopoietic lineage origin using Notch ligand, iPSCs are amplified for 6 days, followed by embryoid body formation, which takes about 8 days. The cells are further cultured for about 5 days to enable HSCs to develop from the CD34+-hematopoietic endothelial cells from which they are derived. The HSCs are then cultured in a medium that promotes differentiation into lymphohematopoietic cells such as the B cell lineage described herein.

[0112] 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 definitive hematopoietic endothelium into multipotent progenitors (MPP), common lymphoid progenitors (CLP), common lymphoid 2 progenitors (LCA-2), early pro-B cells, late pro-B cells, pre-B cells, and immature B cell lineages, and optionally, (ii) contacting lymphohematopoietic lineage cells derived from pluripotent stem cells with a composition comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL3, IL7, and IL15 to initiate the differentiation of the lymphohematopoietic lineage, wherein the culture medium is free of one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor.

[0113] 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 hematopoietic stem or progenitor cells. Notch receptors include Notch-1, Notch-2, Notch-3, and Notch-4. Notch ligands typically 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), SFIP3 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.

[0114] As used herein, "Notch ligand" also includes full-length (native), partial (truncated forms), 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 native Notch ligand. Peptides that mimic Notch ligands are also included. 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 canonical ligands 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, which functions independently of Notch signaling. The C. elegans DSL ligand lacks the DOS motif but has been proposed to cooperate with ligands containing only DOS to activate Notch signaling.

[0115] In some embodiments, the Notch ligand is an anti-Notch (agonist) antibody that can bind 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.

[0116] In some embodiments, the Notch ligand is a delta family Notch ligand. In some embodiments, the delta family ligand is 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 techniques.

[0117] 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.

[0118] 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 naive CD3 + CD8 + and CD3 + CD4 + T cells or B cell lineages. In some embodiments, the artificial thymic organoid comprises DLL4 and BMP2, or functional fragments thereof.

[0119] 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 DLL4 ligands as broad-spectrum modulators of Notch signaling , Nature Chemical Biology (2022).

[0120] 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 US 2020 / 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, iron dextran, 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 polyol, poloxamer, poly(uronic acid), poly(anhydride), poly(vinylpyrrolidone), and any combination thereof. In some embodiments, the scaffold includes pores with diameters between about 1 pm and 100 pm.

[0121] 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 enzymatically conjugated 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.

[0122] 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 together 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 that provides 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 combinations thereof are in contact with the culture conditions, providing topographical patterns and / or textures (e.g., roughness) that are conducive to cell differentiation and / or expansion.

[0123] In various aspects and embodiments, the present disclosure provides a method for obtaining a culture platform for B cell lineage (as described) or its progenitors. In various embodiments, the method comprises contacting cells (e.g., CD34+ cells from EBs generated as described herein) with an agonist of a mechanosensitive receptor or mechanosensitive channel that increases the activity or expression of Dnmt3b in an effective amount. In some embodiments, the mechanosensitive receptor is Piezol. Exemplary representatives of Piezol agonists include Yoda1, single-stranded (ss) RNA (e.g., ssRNA40), Jedi1, and Jedi2. In some embodiments, the mechanosensitive receptor is Trpv4. An exemplary Trpv4 agonist is GSK1016790A. The culture medium may comprise one or more of 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). The correspondingly generated HSC population (and which may be enriched for CD34+ cells) is cultured in the presence of cytokines, growth factors, and / or small molecules that promote the formation of lymphocytes, including B cell lineage. For example, such cytokines and growth factors may include a combination of IL-3, IL-7, IL-15, SCF, and FLT-3L. In some embodiments, particularly in the later stages of culture, IL-3 may be excluded. For example, CD19+ cells may be cultured with IL-7, SCF, and Flt3L. Other methods known in the art for generating B cell lineage may be employed. B cells may be activated in vitro using cytokines and factors such as IL-4, IL-5, IL-6, and CD40 ligand.

[0124] In some embodiments, the B cell lineage expresses a chimeric antigen receptor (CAR) based on gene editing of iPSCs, embryoid bodies, hCD34+ cells, or B cell progenitors or lineages. In some embodiments, the B cell lineage expresses CAR via mRNA expression. Additionally, or optionally, the B CAR may be engineered to express cytokines (e.g., IL-4, IL-6, IL-15, etc. or interferons) to make the B cell-CAR more effective in targeting tumor or infected cells.

[0125] In non-limiting examples, B cells or progenitors can be efficiently transduced by vectors such as, but not limited to, retroviral or non-integrating viral vectors (e.g., adenovirus, adeno-associated virus, integrase-deficient lentivirus, poxvirus) or non-viral vectors (e.g., plasmid vectors, artificial chromosomes) or episomal or episomal hybrid vectors (see, e.g., 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)) carrying a first, second, third, fourth or fifth generation CAR. Each of the above references targeting tumor antigens is hereby incorporated by reference in its entirety).

[0126] The CAR is designed to enhance the ability of the cell to recognize, bind, and kill target cells such as tumor cells or virus-infected cells or tissues. In some embodiments, the CAR enhances the ability of B cells to recognize target cells. In some embodiments, the CAR enhances the activity of B cells, e.g., via B cell activation through antigen presentation, co-stimulation, and cytokine production. In some embodiments, the CAR-B cells target the following tumors or tumor antigens:

[0127] (i) human epidermal growth factor receptor 2 (HER2) - ovarian cancer, breast cancer, glioblastoma, colon cancer, osteosarcoma, and medulloblastoma;

[0128] (ii) epidermal growth factor receptor (EGFR) - non-small cell lung cancer, epithelial cancer, and glioma;

[0129] (iii) mesothelin - mesothelioma, ovarian cancer, and pancreatic adenocarcinoma;

[0130] (iv) prostate-specific membrane antigen (PSMA) - prostate cancer;

[0131] (v) carcinoembryonic antigen (CEA) - pancreatic adenocarcinoma, breast cancer, and colorectal cancer;

[0132] (vi) Glypican-3 - hepatocellular carcinoma;

[0133] (vii) Epidermal growth factor receptor variant III (EGFRvIII) - glioblastoma;

[0134] (viii) Disialoganglioside 2 (GD2) - neuroblastoma and melanoma;

[0135] (ix) Carbonic anhydrase IX (CAIX) - renal cell carcinoma;

[0136] (x) Interleukin-13Ra2 - glioma;

[0137] (xi) Fibroblast activation protein (FAP) - malignant pleural mesothelioma;

[0138] (xii) L1 cell adhesion molecule (L1-CAM) - neuroblastoma, melanoma and ovarian cancer;

[0139] (xiii) Cancer antigen 125 (CA 125) - epithelial ovarian cancer;

[0140] (xiv) Cluster of differentiation 133 (CD 133) - glioblastoma and cholangiocarcinoma, adenocarcinoma;

[0141] (xv) Cancer / testis antigen 1B (CTAG1B) - melanoma and ovarian cancer;

[0142] (xvi) Mucin 1 - seminal vesicle carcinoma;

[0143] (xvii) Folate receptor-a (FR-a) - ovarian cancer;

[0144] (xviii) A growth factor receptor selected from one or more of ErbB1, ErbB2, ErbB3 or ErbB4, IGF1R, IGF2R, TβRI-II, VEGFR1, VEGFR2, VEGFR3, PDGFR(α / β) or FGFR1 to 4.

[0145] Thus, in some aspects and embodiments of the present invention, genetically modified B cell lines or their precursors or progeny are 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), either alone or in combination with additional components from second or third generation CAR constructs (such as CD28, CD134, CD137, and / or ICOS).

[0146] In some embodiments, the CAR comprises at least one domain that inhibits anti - autoimmune or phagocytic signaling in B cells (e.g., extracellular domain, transmembrane domain, and / or intracellular domain). In some embodiments, the CAR improves the effector activity of B cells, for example, by inhibiting CD47 and / or SIRPα activity, relative to the same type of cells without the CAR. In some embodiments, the CAR acts as a dominant - negative receptor (e.g., a CD47 sink) by binding to CD47 and inhibiting SIRPα activity.

[0147] In some embodiments, relative to unmodified B cells, CAR - modified B cells exhibit an increase in the production of one or more inflammatory cytokines. The one or more inflammatory cytokines may 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.

[0148] On the one hand, the present disclosure provides a cell composition comprising a cell population (e.g., a B cell lineage population). The cell compositions of the present disclosure (e.g., prepared according to the present disclosure) may further comprise a pharmaceutically acceptable excipient or carrier. Such excipient or carrier solutions may also contain buffers, diluents, and other suitable additives. A buffer refers to a solution or liquid whose chemical composition 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 may comprise an excipient suitable for intravenous infusion or other routes of administration, and the composition may comprise a suitable cryoprotectant. An exemplary carrier is DMSO (e.g., about 10% DMSO). Other carriers may include dimethoxyethane (DME), N,N-dimethylformamide (DMF), or dimethylacetamide, including mixtures or combinations thereof. The cell composition may be provided in an implantable device (e.g., a scaffold), or in a bag, or in a vial, test tube, or container in a suitable volume and stored frozen until use.

[0149] In various embodiments, the B cells are derived from HLA-edited iPSCs as described. For example, in some embodiments, the B cells are HLA-A neg , homozygous for both HLA-B and HLA-C, HLA-DPB1 neg and HLA-DQB1 neg . In some embodiments, the B cells are further homozygous for HLA-DRB1.

[0150] In some embodiments, the cell population is a progenitor B cell population that is capable of engrafting in the thymus, spleen, or secondary lymphoid organs upon administration to a subject in need. In various embodiments, a composition for cell therapy is prepared that comprises a cell population and a pharmaceutically acceptable excipient. The pharmaceutical composition may 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 that comprises cells at about 100,000 to about 400,000 cells per kilogram (e.g., about 200,000 cells / kg). In other embodiments, about 10 5 to about 5×10 5 cells (e.g., about 2.5×10 5 cells / kg), or about 10 6 to about 5×10 6 cells (e.g., about 2.5×10 6 cells / kg), or about 5×10 6 to about 10 7 cells (e.g., about 5×10 6 cells / kg), or about 10 7 to about 10 8 cells (e.g., about 5×10 7 cells / kg), or about 10 8 to about 10 9 cells (e.g., about 5×10 8 cells / kg), or about 10 9 to about 10 10 cells, or about 10 10 to about 10 11 or about 10 11 to about 10 12 cells, or about 10 12 to about 10 13 cells, or about 10 13 to about 10 14 cells are administered.

[0151] The pharmaceutical composition may also contain additional therapeutic agents for treating a particular targeted disorder. For example, the pharmaceutical composition may also comprise 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 effect the advantages of the treatment methods disclosed herein, namely, to provide improved therapeutic efficacy and reduced systemic toxicity.

[0152] In other aspects, the present invention provides a method of cell therapy comprising administering to a human subject in need thereof a 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, 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, lupus erythematosus systemicus, certain forms of thyroiditis, certain forms of uveitis, vitiligo, granulomatosis with polyangiitis (Wegener). Blood 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 bone marrow failure syndromes and certain genetic diseases (e.g., genetic disorders affecting the immune system). In some embodiments, the subject has cancer, such as 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.

[0153] The cell population can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the particular disease or disorder being treated, the particular mammal being treated (e.g., 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 dosing schedule, and other factors known to the physician. The therapeutically effective amount of the cells to be administered will be governed by such considerations.

[0154] Other compounds, 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)) can be administered together with the cells. Combinatorial administration includes co-administration using separate formulations or a single pharmaceutical formulation and sequential administration in either order, where preferably there is a period of time during which two (or all) of the active agents exert their biological activities simultaneously.

[0155] Regarding the use of HLA-edited B cells (e.g., derived from gene-edited iPSCs), a subject can be matched at the remaining HLA loci, such as one or more (or all) of HLA-B, HLA-C, and HLA-DRB1.

[0156] In certain embodiments, prior to therapy, a patient can undergo lymphodepleting chemotherapy (or other known methods) using chemotherapeutic agents such as fludarabine or cyclophosphamide.

[0157] As used herein, the term “about” means ±10% of the relevant value.

[0158] Certain aspects and embodiments of the present disclosure are further described with reference to the following examples.

[0159] Examples

[0160] 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.

[0161] 方法

[0162] iPSCs were developed from hCD34+ cells by episomal reprogramming known in the art and essentially as described by Yu, et al. Induced pluripotentstemcell lines derivedfromhuman somaticcells , Science 318, 1917 - 1920, (2007); and J. Yu, et al. Humaninduced pluripotent stem cells free of vector and transgene sequences . Science 324, 797 - 801, (2009). Embryoid body and hematopoietic endothelial differentiation were essentially as described in the following references: R. Sugimura, et al., Haematopoietic stem and progenitor ce lls from human pluripotent stem cells . Nature 545, 432 - 438, (2017); C. M. Sturgeon, et al.,Wnt signaling controls the specification of definitive and primitive hematopoiesis from 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 of vector and transgene sequences . As described in Science 324, 797 - 801, (2009).

[0163] Briefly, hiPSCs were 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 were seeded in 10 - cm dishes (EZSPHERE or low - attachment plates) for EB formation. On day 1, bFGF and BMP4 were added to the medium. On day 2, the medium was replaced with a medium containing SB431542, CHIR99021, bFGF, and BMP4. On day 4, the cell medium was replaced with a medium supplemented with VEGF and bFGF. On day 6, the cell medium was replaced with a medium supplemented with bFGF, VEGF, interleukin (IL) - 6, IGF - 1, IL - 11, SCF, and EPO. The cells were maintained in an incubator with 5% CO2, 5% O2, and 95% humidity. To harvest CD34+ cells, the EBs were dissociated on day 8, the cells were filtered through a 70 - μm filter, and CD34+ cells were isolated by CD34 magnetic bead staining.

[0164] Results

[0165] An adenoviral vector containing ETV2 and GFP sequences under the control of the EF1A promoter was used to transduce induced pluripotent stem cells (iPSCs). After transduction, approximately 45% of the iPSC culture was observed to be GFP - positive, confirming ETV2 overexpression (ETV2 - OE). Further observations showed that ETV2 - OE in iPSC cells retained the pluripotent characteristics of iPSCs, as shown by the expression of the stem cell marker TRA - 1 - 60 ( Figure 1 ). Figure 1 FACS plots showing the transduction efficiency of iPSCs overexpressing ETV2 and GFP sequences with an adenoviral vector.

[0166] 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 shows a representative flow cytometry analysis of hematopoietic endothelial cells (defined here as CD235a-CD34+CD31+), and relative quantification indicated that ETV2-OE enhanced the formation of hematopoietic endothelial cells compared to the control.

[0167] In addition, the results showed that ETV2-OE enhanced the formation of CD34 + cells ( Figure 3 ). Figure 3 Shows a representative flow cytometry analysis of CD34+ cells, and relative quantification indicated that ETV2-OE enhanced the formation of CD34+ cells.

[0168] 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.

[0169] Example 2 - iPSC-derived HSCs generated using Piezo1 activation undergo T cell differentiation similar to bone marrow-derived HSCs.

[0170] Methods

[0171] To analyze EHT, EB-derived CD34+ cells 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 culture. After 4 to 7 days, the cells were harvested for analysis.

[0172] 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 culture between days 5 and 7 for further hematopoietic lineage differentiation.

[0173] CD34+ cells harvested from the EHT culture between days 5 and 7 (or a total of days 13 to 21 from iPSC differentiation) were seeded into a 48-well plate pre-coated with rhDL4 and recombinant human fibrin fragment. T-lineage differentiation was induced in a medium containing aMEM, FBS, ITS-G, 2BME, ascorbic acid-2-phosphate, Glutamax, rhSCF, rhTPO, rhIL7, FLT3L, rhSDF-1a and SB203580.

[0174] From day 2 to day 6, 80% of the medium was replaced every other day. On D7, the cells were transferred to a new coated plate and the presence of pro-T cells (CD34+CD7+CD5+ / -) was analyzed.

[0175] From day 8 to day 13, 80% of the medium was replaced every other day. On D14, 100,000 cells / well were transferred to a new coated plate and the presence of pre-T cells (CD34-CD7+CD5+ / -) in the cells was analyzed.

[0176] From day 15 to day 20, 80% of the medium was replaced every other day. The cells were harvested on D21 and analyzed for CD3, CD8, CD5, CD7, TCRab expression by FACS as a surrogate for T cells, and / or activated with CD3 / CD28 beads to evaluate their functional properties.

[0177] After 21 days of differentiation, the cells were collected and re-seeded at approximately 80,000 cells into a new 96-well culture plate in RPMI 1640 (without L-glutamine; without phenol red) plus FBS, L-glutamine, IL-2, and then activated with 1:1 CD3 / CD28 beads. After activation with CD3 / CD28 beads for 72 hours, the cells were analyzed for CD3, CD69, CD25 expression by FACS and IFN-γ expression was analyzed using RT-qPCR. The supernatant was analyzed by ELISA.

[0178] Results

[0179] Figure 4A and Figure 4B showed that iPSC-derived HSCs of EHT from D8 CD34+ cells (activated with Piezo1 in this example) underwent pro-T cell differentiation similar to bone marrow (BM)-HSCs. In addition, Figure 5A and Figure 5B showed that iPSC-derived HSCs generated from EHT of D8 CD34+ cells (activated with Piezo1 in this example) underwent T cell differentiation and could be activated by CD3 / CD28 beads similar to BM-HSCs. Figure 6Shown is that iPSC-derived HSCs generated by EHT of D8 CD34+ cells (activated with Piezo1 in this example) can differentiate into functional T cells, as demonstrated by the expression of INFγ upon stimulation with CD3 / CD28 beads. Collectively, these results indicate that EHT of D8 CD34+ cells enhances the ability to further differentiate into hematopoietic lineages in vitro.

[0180] Example 3 – Assessment of off-target editing in HLA-edited HSCs

[0181] HLA typing of HSC clones that have undergone HLA editing (e.g., triple knockout) is performed to check for unwanted editing and to ensure that no major editing events, such as deletions, have occurred in other regions of chromosome 6. Sequencing methods and analyses are performed to evaluate the extent of gRNA off-target activity, and gRNAs representing a low risk of affecting non-target HLA genes are selected.

[0182] Sequencing is performed using in situ break labeling in fixed and permeabilized cells by ligating a full-length P5 sequencing adapter to the ends of the prepared DSBs. Genomic DNA is extracted, fragmented, end-prepared, and ligated using a chemically modified semi-functional P7 adapter. The resulting DNA library contains a mixture of functional DSB-labeled fragments (P5:P7) and non-functional genomic DNA fragments (P7:P7). Subsequently, DNA sequencing of the DNA library enriched for DNA-labeled fragments is performed, removing all foreign, non-functional DNA. Since library preparation does not contain PCR, each sequencing read obtained corresponds to a single labeled DSB end from a cell. This generates 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.

[0183] Table 1 below summarizes the results of the editing strategies for two representative clones relative to wild-type cells.

[0184] Table 1: Clonal HSC HLA knockout.

[0185]

[0186]

[0187] Table 2 provides non-limiting examples of gRNAs used in the experiments, which can be used to knock out the expression of the specified HLA genes and haplotypes.

[0188] Table 2: Exemplary gRNA sequences

[0189]

[0190]

[0191] The results showed 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.

[0192] These results were confirmed by phenotypic analysis of HLA-edited clones by FACS and immunofluorescence. As Figure 9 shown in Figures 6A and 9B, 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 (gHSCs). Specific expression of HLA-A by immunofluorescence confirmed that HLA-A was not expressed in HLA-edited cells, corroborating the finding that the gene editing strategy was only successful in deleting the HLA-A gene. Specifically, Figure 7A it was shown 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, despite the HLA-A deletion, other class I molecules such as HLA-B and C were expressed and were not affected by the gene editing strategy.

[0193] To confirm the HLA-A gene deletion, specific expression of HLA-A was analyzed by immunofluorescence. As Figure 7B seen, HLA-A was not expressed in HLA-edited clones, indicating that the gene editing strategy was efficient only in specifically deleting the HLA-A gene. Such retention of all class I expression and HLA-A deletion would facilitate patient matching while avoiding NK cell-mediated rejection.

[0194] Example 4 - Evaluation of the pluripotency and immunocompatibility of HLA-edited HSCs

[0195] Evaluate the ability of HLA-edited cells to retain pluripotency. As Figure 8 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.

[0196] HLA class I molecules are expressed on the surface of all nucleated cells, and if there is a mismatch of HLA class I molecules between the donor and the recipient, the cells can be recognized and killed by CD8+ T cells. In addition, HLA mismatch may lead to cytokine release syndrome (CRS) and graft-versus-host disease (GVHD). In contrast, 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 deletion of HLA-A 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 the grafts of HLA-edited and wild-type HSCs (gHSCs).

[0197] Wild-type (i.e., gHSCs) 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 degree of cytotoxicity mediated by HLA-null and HLA-mismatched PBMCs, respectively. Figure 9 Results of PBMC-mediated cytotoxicity assays in co-cultures measured by Annexin V staining are shown. The results show that 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.

[0198] In summary, the immunocompatibility results show that CD8+ T cells present in the PBMC samples are responsible for killing cells with mismatched HLA molecules (WT and CIITA KO), while NK cells present in the PBMCs 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 expression of HLA class I molecules is largely retained).

[0199] Example 5 - Evaluation of the in vivo engraftment potential of HLA-edited HSCs

[0200] To evaluate the transplantation 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 Figure 10 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.

[0201] Example 6 - Differentiation of HLA-Edited HSCs into CD4+ / CD8+ T Cells

[0202] 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, HLA class I molecules are expressed on the surface of all nucleated cells and display 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. Thus, studies were conducted to determine whether the deletion of HLA-A affects class I peptide presentation by edited HSCs. As Figure 11A and 11B shown, immunopeptidome analysis revealed that the deletion of HLA-A does not affect the overall presentation of class I peptides. When compared to wild-type HSCs (gHSCs), HLA-A-edited cells showed comparable peptide and protein presentation. Additionally, as Figure 12A and 12B 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 these cells (and the lineages derived from them) retain their ability to present a broad spectrum of class I and class II peptides despite the deletion of HLA-A, HLA-DQ, and HLA-DP molecules.

[0203] Example 7 - In Vivo Testing of Antigen-Mediated Immune Responses.

[0204] Figure 13It 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 APCs. CD4+ T cells recognize the peptide-MHC on the antigen-presenting cell (APC). When challenged with antigen, CD4+ helper T cells are activated, and cytokines recruit macrophages and other immune cells, which induces tissue swelling.

[0205] 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, the 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 micrometer caliper.

[0206] As Figure 14A and 14B 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 mice transplanted with both WT (unedited HSCs) and HLA-edited HSCs.

[0207] Example 8 - Evaluation of the differentiation and maturation of HSC-derived T cells (pro-T cells)

[0208] Next, the ability of HSC-derived T cells (pro-T cells) to differentiate into mature T cells was tested. After a 35-day differentiation period, the presence of CD4+, CD8+, and αβ+ T cell populations in the pro-T cells was evaluated by cell sorting. As Figure 15 shown, the pro-T cells differentiated more efficiently into CD4+, CD8+, and αβ+ T cells than bone marrow (BM)-derived CD34+ cells and CD34+ cells derived from embryoid bodies (EBs).

[0209] Next, to test the functional characteristics, each T cell population was co-cultured with a CD19+ lymphoma cell line and an anti-CD3 / CD-19 bispecific antibody. In this experimental model, the bispecific antibody acts simultaneously on the CD3 receptor on the T cells and the CD19 cell surface receptor on the lymphoma cells, thus triggering T cell activation. The degree of activation was evaluated by measuring the subsequent T cell-mediated cytotoxicity compared to a pan-T cell control. As Figure 16As shown, compared with BM CD34+ T cells and EB CD34+ T cells, naive T cells showed a statistically significant superiority in cytotoxicity.

[0210] Example 9 – Evaluation of the properties of HSCs developing into naive T cells.

[0211] The ability of HSCs to develop into naive T cells was evaluated by measuring the CD34-CD7+ markers on naive T cells. As Figure 17 shown, FACS analysis showed that, compared with bone marrow-derived CD34+ cells or EB-derived CD34+ cells, the HSCs generated according to the present disclosure successfully differentiated into CD34-CD7+ naive T cells.

[0212] Next, the expression of T cell-specific transcription factors and thymic seeding molecules was measured. Figure 18A It was shown that the expression of TCF7 increased in the naive T cells derived from the HSCs of the present disclosure, and Figure 18B it was shown that the expression of CCR7 increased. Figure 19A It was shown that the naive T cells derived from HSCs seeded and differentiated in the thymus. Figure 19B Shown is the FACS analysis of the CD3 cell population gated on the CD45+ cell population, which shows that the naive T cells derived from HSCs have excellent seeding and differentiation potential in the thymus. The naive T cells of this example were prepared from HSCs using Piezo1 activation as described above.

[0213] As Figure 20 shown, the in vitro activation of T cells derived from HSCs was also measured. Figure 20 The top inset shows the FACS analysis of activated T cells from different sources, including the HSCs of the present disclosure (e.g., prepared using Piezo1 activation). The T cells prepared from the HSCs of the present disclosure showed comparable or superior activation, as measured by increased CD107 expression. The bottom inset shows Dynabeads activation, in which the activated T cells expressed inflammatory cytokines. According to the present disclosure, the T cells derived from HSCs (e.g., prepared using Piezo1 activation) expressed higher levels of inflammatory cytokines, as exemplified by the expression levels of TNF-α and interferon γ.

[0214] Example 10: HLA-edited HSCs differentiate into hematopoietic lineages, namely naive monocytes / macrophages

[0215] Experiments were conducted to determine whether HLA deficiency 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 monocytes / macrophage lineages comparable to WT (non-HLA-edited) HSCs, as measured by their CD11b+-CD14+ expression ( Figure 21A ). Additionally, the CD11b+-CD14+ gated population showed equivalent HLA-I and HLA-II expression ( Figure 21B ), 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 Figure 22A . It was found that the deletion of HLA-DQB1 and HLA-DPB1 did not affect the expression of other HLA class II molecules ( Figure 22B ). For example, HLA-DR had comparable expression in both WT and HLA-edited cells ( Figure 22C ). In Figure 22B and 22C , CIITA-KO was used as a positive control.

[0217] Example 11: Differentiation of HLA-edited HSCs into proplatelets

[0218] It has been determined that HLA-edited HSCs are able to differentiate into megakaryocytes (MK) and further differentiate into platelets. Differentiation was compared for bone marrow (BM)-derived CD34+ cells and iPSC-CD34+ cells. As Figure 23 shown, HLA-edited HSCs showed a statistically significant increase in platelet content compared to BM CD34+ and iPSC-34+ cell populations. Thus, HLA-edited HSCs can differentiate into megakaryocytes (MK), and megakaryocytes can further support differentiation into platelets.

[0219] Example 12 - Evaluation of degranulation and cytotoxicity of immune-compatible HSC-derived NK cells

[0220] Next, the ability of triple-knockout HSCs to differentiate into NK cells that retain their degranulation and cytotoxicity capabilities was evaluated. As Figure 24As shown, HSCs effectively differentiated into NK cells, as determined by fluorescence-activated cell sorting (FACS) experiments (gating based on the expression of the known NK cell surface marker CD56). HSCs demonstrated a differentiation capacity at least comparable to that of CD34+ BM and iPSC-EB CD34+ cell populations. To measure the ability of HSC-derived NK cells to effectively kill tumor cells, the experimental protocol shown in Figure 25A was performed. HSC-derived NK cells were co-cultured with K562 HLA-null cells for 3.5 hours. K562 HLA-null cells are a human erythroleukemia cell line derived from the pleural effusion of a patient with chronic myeloid leukemia. These cells express ligands for aNKR and they lack HLA cell surface expression, and also contribute to NK cell activation by eliminating negative signaling through iNKR. Thus, these HLA-null cell lines have the potential to induce distinct functional characteristics in NK cells and their subsets. After co-culture, FACS and annexin V staining, as well as cytotoxicity assays, were used to measure the extent of NK cell degranulation. As shown in Figure 25B , annexin V staining indicated that HSC-derived NK cells exhibited a higher degree of activation from HLA-null K562 cells compared to CD34+ BM and iPSC-EB CD34+ cells. This was confirmed by the results of the cytotoxicity assays, as shown in Figure 25C .

[0221] References

[0222] 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).

[0223] 2. Brauer, P. M., Singh, J., Xhiku, S. & -Pflücker, J. C. T Cell Genesis: In Vitro Veritas Est? Trends Immunol 37, 889–901 (2016).

[0224] 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).

[0225] 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).

[0226] 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).

[0227] 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).

[0228] 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).

[0229] 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).

[0230] 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).

[0231] 10. Guo, R. et al. Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors. Cell Research 30, 21–33 (2020).

[0232] 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).

[0233] 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 B 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 HSCs and / or HSPCs into a population of progenitor B cells or a population of B cells.

2. The method according to claim 1, wherein the PSC population is a human iPSC population derived from lymphocytes, 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+ cells isolated from peripheral blood.

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

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

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

7. The method according to any one of claims 2 to 4, wherein the iPSC is 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 governing the expression or presentation ability of HLA or MHC.

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

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

10. The method according to any one of claims 2 to 9, wherein the iPSCs are gene-edited to be homozygous for both HLA-A neg , HLA-B and HLA-C, 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 governing 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 CD34+ enrichment and endothelial-to-hematopoietic cell transition are induced on days 8 to 15 of iPSC differentiation.

13. The method according to any one of claims 1 to 12, wherein the CD34+-enriched population is cultured in a medium containing Y-27632, TPO, IL-3, SCF, IL-6, IL-11, IGF-1, VEGF, bFGF, BMP4, and FLT3.

14. The method according to claim 12 or 13, wherein the endothelial-to-hematopoietic cell transition generates a population of HSCs, the population of HSCs comprising one or more of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells, and hematopoietic stem cell progenitors.

15. The method according to any one of claims 12 to 14, wherein CD34+ cells are harvested from a culture undergoing an endothelial-to-hematopoietic cell transition, including harvesting CD34+ floating cells and / or adherent cells.

16. The method according to any one of claims 1 to 15, wherein the HSC population comprises long-term hematopoietic stem cells (LT-HSC).

17. The method according to any one of claims 1 to 16, wherein the induction of the endothelial-to-hematopoietic cell transition includes increasing the expression or activity of dnmt3b.

18. The method according to claim 17, wherein the induction of the endothelial-to-hematopoietic cell transition includes applying cyclic stretching to the CD34-enriched cells.

19. The method according to claim 18, wherein the cyclic stretching is 2D, 3D, or 4D cyclic stretching.

20. The method according to any one of claims 1 to 16, wherein the induction of the endothelial-to-hematopoietic cell transition includes Piezo1 activation.

21. The method according to claim 20, 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, ssRNA40, Jedi1, Jedi2, or an analogue or derivative thereof.

22. The method according to any one of claims 1 to 16, wherein the induction of the endothelial-to-hematopoietic cell transition includes Trpv4 activation.

23. The method according to claim 22, 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 an analogue or derivative thereof.

24. The method according to any one of claims 1 to 23, wherein the B cell lineage is selected from multipotent progenitors (MPP), common lymphoid progenitors (CLP), common lymphoid 2 progenitors (LCA-2), early pro-B cells, late pro-B cells, pre-B cells, and immature B cells.

25. The method according to any one of claims 1 to 24, wherein the B cell lineage can differentiate into one or more lineages having a phenotype consistent with transitional B cells, regulatory B cells, marginal zone B cells, follicular B cells, activated B cells, memory B cells, or plasma B cells, or a combination thereof.

26. The method according to claim 24 or 25, wherein the B cell lineage expresses a chimeric antigen receptor (CAR).

27. The method according to claim 26, wherein the B cell lineage immune cells of the CAR-modified immune cells are selected from one or more of CAR-transitional B cells, CAR-regulatory B cells, CAR-marginal zone B cells, or CAR-follicular B cells, or CAR-activated B cells, CAR-memory B cells, or CAR-plasma B cells.

28. A population of B cell lineage cells or a pharmaceutically acceptable composition thereof, produced by the method according to any one of claims 1 to 27.

29. A B cell population or a pharmaceutically acceptable composition thereof, wherein the B cell population is HLA-A neg homozygous for both HLA-B and HLA-C, HLA-DPB1 neg and HLA-DQB1 neg and optionally further homozygous for HLA-DRB1.

30. A method for cell therapy, the method comprising administering to a human subject in need thereof the population of B cell lineage cells or a pharmaceutically acceptable composition thereof according to claim 28 or claim 29.

31. The method according to claim 30, 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.

32. The method according to claim 31, wherein the subject has cancer, which is optionally a solid tumor.

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