Compositions and methods for differentiating and expanding B lineage cells

Abstract

Media, kits, and methods for directed differentiation of cells into B-cell lineage are disclosed. The disclosed differentiation procedures can take primary cells or pluripotent stem cell-derived cells through one or more intermediate cell populations to result in B-lineage cells using one or more stage-specific media formulations. Thus, media and supplements for carrying out the directed differentiation workflow can be included in a kit that includes one or more basal media and one or more supplements added thereto.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 182,054, filed April 30, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to cell culture applications, more particularly to cell culture applications using hematopoietic cells, and even more particularly to cell culture applications related to one or more population(s) of B cells. [Background technology]

[0003] Mammalian blood is composed of various cell types, including lymphocytes, platelets, red blood cells, and their direct and indirect precursors. Leukocytes, sometimes called white blood cells, function in the host's immune system. Leukocytes can be further subdivided into B cells, T cells, NK cells, monocytes, macrophages, dendritic cells, eosinophils, basophils, and neutrophils. Each of these leukocytes performs a specific function in the host's immune system.

[0004] B cells, like other blood cells, are derived from hematopoietic stem / progenitor cells (HSPCs) that are capable of self-renewal and differentiation into each blood lineage. B cells are central components of humoral immunity and secrete antibodies upon binding to antigens (via the B cell receptor expressed on their surface).

[0005] In vivo, mammalian B cells develop in the bone marrow, beginning as HSPCs and progressing through various developmental stages, including pro-B cells, pre-B cells, and immature B cells. Immature B cells mature into memory B cells in secondary lymphoid organs (e.g., spleen, thymus, etc.) and then into plasmablasts and plasma cells (i.e., antibody-producing cells) in either secondary lymphoid organs or bone marrow. Recapitulating these developmental steps in vitro or ex vivo has met with limited success. In particular, it has not been possible to recapitulate these developmental steps in serum-free and feeder-free culture systems using tissue-derived cells or pluripotent stem cells ("PSCs") as source material.

[0006] Both primary tissue-derived cells and PSCs offer the opportunity to generate large, homogenous, customizable populations of B-lineage cells, particularly those suitable for clinical applications. Differentiated PSCs also enable genetic engineering methods that facilitate disease modeling or cell therapy applications.

[0007] B cells are of intense research and therapeutic interest given their role in sensing antigens in the environment and their ability to secrete large amounts of antibodies to neutralize targets upon stimulation. Thus, there is a need for efficient means of obtaining immature and mature B lineage cells in culture from precursor populations, whether derived from PSCs or appropriate precursors isolated from umbilical cord blood or bone marrow. Summary of the Invention

[0008] The present disclosure relates to medium compositions and / or supplements added to medium, and methods for the culture / differentiation of hematopoietic stem / progenitor cells (HSPCs). More specifically, the present disclosure relates to methods for the stepwise differentiation of HSPCs into various B cell lineages using stage-specific medium and / or supplements added to basal medium.

[0009] In one aspect of the present disclosure, CD34 +Provided is a directed differentiation method for preparing a population of hematopoietic stem or progenitor cells (HSPCs), comprising contacting a population of HSPCs with an induction medium comprising a basal medium, at least one of stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L), and at least one other cytokine, and culturing the population of HSPCs in the induction medium under serum-free conditions to obtain a population of B cell precursors.

[0010] In one embodiment, the at least one other cytokine is one or more of IL-3, IL-6, or IL-7. In one embodiment, the at least one other cytokine is one or more of IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, or IL-21.

[0011] In one embodiment, a population of HSPCs is enriched from umbilical cord blood or bone marrow, or differentiated from pluripotent stem cells (PSCs).

[0012] In one embodiment, the population of B cell precursors expresses one or both of CD10 or CD19.

[0013] In one embodiment, the induction medium contains either SCF or TPO.

[0014] In one embodiment, the method may further comprise contacting the population of B cell precursors with a differentiation medium and culturing the population of B cell precursors in the differentiation medium under serum-free conditions. + This may further include obtaining a population of B-lineage cells.

[0015] In one embodiment, the method provides for the production of more CD19 than after culturing the population of HSPCs in induction medium. + It may further comprise obtaining B lineage cells.

[0016] In one embodiment, CD19 +At least some B lineage cells express IgM + It is a cell.

[0017] In one embodiment, the differentiation medium comprises a basal medium, at least one of SCF, TPO, and FLT3L, and at least one other cytokine.

[0018] In one embodiment, the method comprises administering to a subject a subject, comprising administering to a subject a subject, ... + contacting a population of B lineage cells with a downstream differentiation medium, and + The method may further comprise culturing the population of B-lineage cells under serum-free conditions. In one embodiment, the method results in more IgM cells than after culturing the population of B-cell precursors in differentiation medium. + It may further include obtaining the cells.

[0019] In one embodiment, IgM + At least some of the cells are antibody-secreting cells.

[0020] In one embodiment, the downstream differentiation medium comprises a basal medium, a ligand for human CD40, and at least one other cytokine.

[0021] In one embodiment, the method is carried out under feeder cell-free conditions. In one embodiment, the feeder cell-free conditions include extracellular matrix proteins or cell adhesion molecules. In one embodiment, the feeder cell-free conditions are in the absence of extracellular matrix proteins or cell adhesion molecules that are solubilized or coated on the surface of the culture vessel.

[0022] In one embodiment, the extracellular matrix protein or cell adhesion molecule is solubilized or coated onto the surface of the culture vessel. In one embodiment, the extracellular matrix protein or cell adhesion molecule is fibronectin, vitronectin, laminin, ECM1, SPARC, osteopontin, vascular cell adhesion molecule, immobilized SCF protein, or any combination thereof.

[0023] In another aspect of the present disclosure, there is provided a directed differentiation method for preparing a population of B-lineage cells, comprising contacting a population of B-cell precursors with a differentiation medium comprising a basal medium, at least one of SCF, TPO, and FLT3L, and at least one other cytokine, and culturing the population of B-cell precursors in the differentiation medium under serum-free conditions to obtain a population of B-lineage cells.

[0024] In one embodiment, the at least one other cytokine is one or more of IL-3, IL-6, or IL-7. In one embodiment, the at least one other cytokine is one or more of IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, or IL-21.

[0025] In one embodiment, the population of B cell precursors expresses one or both of CD10 or CD19.

[0026] In one embodiment, the population of B cell precursors is either enriched from umbilical cord blood or bone marrow, or differentiated from pluripotent stem cells (PSCs), CD34 + They are derived from a population of hematopoietic stem or progenitor cells (HSPCs).

[0027] In one embodiment, the population of B lineage cells expresses CD19. In one embodiment, the population of B lineage cells expresses more CD19 than after culturing the population of HSPCs in induction medium to obtain a population of B cell precursors. + Contains cells.

[0028] In one embodiment, the induction medium is serum-free. In one embodiment, the induction medium comprises a basal medium, at least one cytokine, and one or more of SCF, TPO, and FLT3L.

[0029] In one embodiment, CD19 + At least some B lineage cells express IgM + It is a cell.

[0030] In one embodiment, the method may further comprise contacting the population of B-lineage cells with a downstream differentiation medium and culturing the population of B-lineage cells in the downstream differentiation medium under serum-free conditions. ... comprise culturing the population of B-cell precursors in the differentiation medium under serum-free conditions. + It may further include obtaining the cells.

[0031] In one embodiment, IgM + At least some of the cells are antibody-secreting cells.

[0032] In one embodiment, the downstream differentiation medium comprises a basal medium, a ligand for human CD40, and at least one other cytokine.

[0033] In one embodiment, the method is carried out under feeder cell-free conditions. In one embodiment, the feeder cell-free conditions include extracellular matrix proteins or cell adhesion molecules. In one embodiment, the feeder cell-free conditions are in the absence of extracellular matrix proteins or cell adhesion molecules that are solubilized or coated on the surface of the culture vessel.

[0034] In one embodiment, the extracellular matrix protein or cell adhesion molecule is solubilized or coated onto the surface of the culture vessel. In one embodiment, the extracellular matrix protein or cell adhesion molecule is fibronectin, vitronectin, laminin, ECM1, SPARC, osteopontin, vascular cell adhesion molecule, immobilized SCF protein, or any combination thereof.

[0035] In another aspect of the present disclosure, a kit for directed differentiation of B-lineage cells is provided, the kit comprising a basal medium and at least one supplement, in one embodiment, the at least one supplement comprises at least one of stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L), and at least one other cytokine.

[0036] In one embodiment, the kit further comprises a second supplement.

[0037] In one embodiment, the second supplement comprises at least one of stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L), and at least one other cytokine.

[0038] In one embodiment, the formulation of at least one supplement is different from the formulation of a second supplement.

[0039] In one embodiment, the kit further comprises a third supplement, hi one embodiment, the third supplement comprises a ligand for human CD40 and at least one other cytokine.

[0040] In one embodiment, the at least one cytokine is one or more of IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, or IL-21.

[0041] In other aspects of the disclosure, B cell precursors, or B lineage cells, or cells downstream of B lineage cells, such as IgM + Media for directed differentiation of cells and / or antibody-secreting cells are provided.

[0042] In one aspect of the present disclosure, a medium for inducing B cell precursors is provided. In one embodiment, the induction medium comprises a basal medium, at least one of stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L), and at least one other cytokine.

[0043] In one aspect of the present disclosure, a medium for differentiation of B-lineage cells is provided. In one embodiment, the differentiation medium comprises a basal medium, at least one of stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L), and at least one other cytokine.

[0044] In one aspect of the present disclosure, a medium for downstream differentiation of B-lineage cells is provided. In one embodiment, the downstream differentiation medium comprises a basal medium, a ligand for human CD40, and at least one other cytokine.

[0045] In one embodiment, the at least one cytokine included in the medium of the present disclosure is one or more of IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, or IL-21.

[0046] For a better understanding of the various embodiments described herein, and to show more clearly how these may be put into practice, reference will now be made, by way of example, to the accompanying drawings illustrating at least one example embodiment, and which are not intended to limit the scope of the teachings described herein. [Brief explanation of the drawings]

[0047] [Figure 1]Representative flow cytometry plots of CD34+ HSPCs enriched from a single human umbilical cord blood donor sample are shown. The enriched population (i.e., "bulk") of CD34+ cells was stained and then sorted for Lin- (Lin included CD3, CD14, CD15, CD16, CD19, CD56, and CD66b) (A). Cells in (A) were further sorted to obtain two more specialized populations of putative B-cell fate progenitors: (B) Lin-CD34+CD38- / lo / midCD10- cells ("population 1" or "pop1") and (B) Lin-CD34+CD38midCD10+ cells ("population 2" or "pop2"). Pop1 cells have multipotency for granulocytic, monocytic, lymphoid, and erythroid progeny, whereas pop2 cells are restricted to the lymphoid lineage. [Figure 2] A bar graph summarizing the results of the induction of B cell precursors from a population of CD34+ HSPCs is shown. Pop2 cells were cultured for 14 days in various induction medium formulations ("SUPPLCTL" baseline medium and formulations lacking the indicated cytokines / growth factors) to test the effect of the absence of selected growth factors or cytokines. After 14 days, the abundance and yield of B cell precursors (A) and CD19+-expressing cells (B) were determined. Results shown are the average of one to three independent experiments. [Figure 3] Bar graphs summarizing the results of B cell precursor induction from a population of CD34+ HSPCs are shown. Pop1 and pop2 cells were separately cultured for 14 days in induction medium excluding TPO but further containing the indicated cytokines, either alone or in combination. For pop2-derived cells, the bar graphs show the overall fold expansion (A), abundance and yield of B cell precursors (B), abundance and yield of CD19+ expressing cells (C), and a sample flow plot analyzing CD10 and CD19 expression (D). For pop1-derived cells, the bar graphs show the overall fold expansion (E), abundance and yield of B cell precursors (F), abundance and yield of CD19+ B lineage cells (G), and a sample flow plot analyzing CD10 and CD19 expression (H). Results shown are the average of two to eight independent experiments. [Figure 4] Bar graphs summarizing the results of differentiation of CD19+ B-lineage cells from a population of CD34+ HSPC-derived B-cell precursors are shown. Pop2 cells were first cultured for 14 days in the control formulation of Figure 2 and then transferred to various differentiation media formulations ("DiM") omitting individual factors and factor combinations as indicated for an additional 14 days. After 28 days of culture, the abundance and yield of B-cell precursors (A), CD19+ B-lineage cells (B), and IgM+ cells (C) among the resulting cells were determined. Results shown are the average of one to three independent experiments. [Figure 5] Figure 1 shows bar graphs comparing the differentiation efficiency of CD34+ HSPCs in media of the present disclosure. Pop1 and pop2 cells were cultured separately in two versions of induction medium for 14 days and then transferred to the differentiation medium formulation of Figure 4 for an additional 14 days. For pop2-derived cells, the bar graphs show the abundance and yield of B cell precursors (A), the abundance and yield of CD19+ B-lineage cells (B), and the abundance and yield of IgM+ cells as a function of the CD19+ cell fraction (C). For pop1 cells, the bar graphs show the abundance and yield of B cell precursors (D), the abundance and yield of CD19+ B-lineage cells (E), and the abundance and yield of IgM+ cells as a function of the CD19+ cell fraction (F). Results shown are the average of four independent experiments. [Figure 6] Figure 1 shows bar graphs summarizing the results of differentiation of CD19+ B-lineage cells from a population of CD34+ HSPC-derived B-cell precursors. Pop1 cells were first cultured for 14 days in the induction medium formulation shown in Figure 2 (but different from that used in Figures 3-5). The resulting B-cell precursors were transferred to the differentiation medium of the present disclosure for an additional 14 days. After 28 days, the abundance and yield of B-cell precursors (A) and CD19+ B-lineage cells (B) were determined. Results shown are the mean ± standard error of one to five independent experiments. [Figure 7]Bar graphs comparing the effect of expansion on B-lineage cell differentiation are shown. Pop1 cells were first cultured in induction medium for 14 days and then transferred to differentiation medium for an additional 14 or 28 days. Bar graphs show the abundance and yield of CD19+ B-lineage cells (A) and the abundance and yield of IgM+ cells as a function of the CD19+ cell fraction (B). Results shown are the mean ± standard error of four independent experiments. [Figure 8] 1 shows a bar graph summarizing the abundance and yield of CD19+ cells at day 28 differentiated using the media formulations and methods of the present disclosure, starting from either bulk CD34+ cells, pop1 CD34+ cells, or pop2 CD34+ cells. Bars represent the mean of at least 23 independent data points. [Figure 9] Results of an experiment to optimize the differentiation efficiency of IgM+ cells from CD19+ B-lineage cells are shown. Pop1 cells or bulk cells were separately cultured in induction medium for 14 days and then transferred to differentiation medium for an additional 14 days. Day 28 cells were then separately cultured in various downstream differentiation medium ("DDM") formulations: a negative control and two formulations containing a human CD40 ligand and various cytokines / growth factors. Day 35 pop1-derived cells were analyzed for the abundance and yield of CD19+ B-lineage cells (A) and IgM+ cells (B) as a function of the CD19+ cell fraction. Results shown are the average of at least four independent experiments. Day 35 pop1-derived cells were also tested for IgM (red / gray) and IgG (blue / black) secretion in ELISPOT assays. Representative images are shown for control cells (Ci) and cells cultured in the indicated downstream differentiation medium (Cii and Ciii), along with the number of antibody-secreting cells generated. Bulk-derived cells on day 35 were analyzed for abundance and yield of CD19+ B-lineage cells (D) and IgM+ cells as a function of the CD19+ cell fraction (E). Results shown are the average of at least three independent experiments. [Figure 10]Figure 1 shows the results of inducing B cell precursors from a population of human PSC-derived CD34+ HSPCs. PSC-derived CD34+ HSPCs were cultured in the disclosed induction medium for 14 days, either in the absence or presence of different coating materials, as indicated. Results shown are the average of at least two independent experiments (A). Day 28 cell populations cultured in the absence or presence of various coating materials were analyzed by flow cytometry for CD10 and CD19 expression (B). PSC-derived or cord blood-derived bulk cells were analyzed by qRT-PCR for EBF1 (C) and PAX5 (D) expression at the indicated time points of the induction / differentiation protocol described herein. Day 28 cell populations were analyzed by flow cytometry for CD10 and CD19 expression, as well as CD19 and CD20 expression (E). Bars represent the mean ± standard error of at least one experiment. [Figure 11] Bar graphs summarizing the effect of various coatings on the induction / differentiation of cord blood-derived CD34+ cells are shown. Pop1 cells were cultured for 14 days in induction medium on the indicated coatings, and the abundance and yield of B cell precursors (A) and CD19-expressing cells (B) were determined. Day 14 cells were cultured for an additional 14 days in differentiation medium on the indicated coatings, and the abundance and yield of CD19+ B-lineage cells (C) and the abundance of IgM+ cells as a function of the CD19+ cell fraction (D) were determined at day 28. Results shown are the average of at least two independent experiments. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present disclosure relates to medium compositions and / or supplements added to medium, and methods for the culture / differentiation of HSPCs. More specifically, the present disclosure relates to the differentiation of HSPCs into various B cell lineages using supplements added to stage-specific medium and / or basal medium.

[0049] As used herein, the term "hematopoietic stem or progenitor cell" or "HSPC" refers to a cell of the hematopoietic lineage that is capable of self-renewal and / or differentiation into more specialized cells of the hematopoietic lineage. In some embodiments, HSPCs may be included in a cell population, and the population of HSPCs may be greater than 50% pure, greater than 60% pure, greater than 70% pure, greater than 80% pure, or greater than 90% pure. HSPCs may be obtained from bone marrow (BM), umbilical cord blood (CB), embryonic to adult peripheral blood (PB), thymus, peripheral lymph nodes, gastrointestinal tract, tonsils, pregnant uterus, liver, spleen, placenta, or any other tissue that contains a localized population of HSPCs. In some embodiments, HSPCs (or a population of HSPCs) are enriched from a tissue source or another cell population containing HSPCs, for example, by immunomagnetic separation or fluorescence-activated cell sorting. HSPCs may also be differentiated from pluripotent stem cells, such as induced pluripotent stem cells, embryonic stem cells, naive stem cells, or elongated stem cells. A hallmark of HSPCs is their expression of the transmembrane phosphoglycoprotein CD34; therefore, HSPCs are CD34 + Human HSPCs are sometimes referred to as HSPCs. Human HSPCs are further defined by the expression of CD45 and CD34, and may be further defined by combinations of markers such as CD38, CD43, CD45RO, CD45RA, CD10, CD49f, CD59, CD90, CD109, CD117, CD133, CD166, HLA-DR, CD201, and integrin-α3, which can be used to distinguish HSPC subpopulations. HSPCs may not express, or may only express low levels of, markers such as glycophorin A, CD3, CD4, CD8, CD14, CD15, CD19, CD20, and CD56, which may characterize more mature blood cells.

[0050] As used herein, the term "pluripotent stem cells" or "PSCs" refers to cells capable of self-renewal and / or differentiation into any cell type of any of the three embryonic germ layers. PSCs, such as embryonic stem cells, may be isolated from blastocysts and subjected to maintenance or differentiation cell culture conditions. PSCs, such as induced pluripotent stem cells, may be derived from any cell type by forced expression of specific pluripotency genes, such as Oct4, Nanog, Sox2, and Klf4. Expression of pluripotency genes can be forced by stably or transiently introducing their coding regions into host cells or by introducing factors that activate endogenous copies of such genes.

[0051] As used herein, the term "B cell precursor" or "B cell precursors" refers to a cell type that is more specialized than an HSPC but is capable of further differentiation into one or more lymphoid cell types, such as B cells. B cell precursors may be direct descendants of HSPCs, whether tissue-derived or PSC-derived, or may be more distant from HSPCs. Furthermore, B cell precursors may differentiate directly into downstream lymphoid cell types, such as B cells, or may undergo one or more additional differentiation steps before becoming B cells. An example of a B cell precursor is a cell that is positive for one of the phenotypic markers CD10 or CD19. In one example, CD10 + B cell precursors are negative for CD19, and such cells may not commit to the B lineage. +B cell precursors are negative for CD10, and such cells may be committed to the B lineage. Other phenotypic markers that may be expressed by B cell precursors include CD20, CD45RA, CD34, CD38, CD161, CD122, CD117, CD127, and / or integrin β7. Additionally, examples of phenotypic markers that may not be expressed by B cell precursors include CD10, CD19, CD20, CD45RA, CD34, CD38, CD161, CD122, CD117, CD127, and / or integrin β7. Unless otherwise specified, a population of B cell precursors herein may refer to a homogeneous population of cells or a heterogeneous population of cells capable of differentiating into one or more downstream cell types. In one embodiment, the B cell precursors may be capable of differentiating into any type of B lineage cell. In one embodiment, the B cell precursors may be negative for CD10, for example, double-positive CD10. + CD19 + They may be further restricted in their differentiation potential so that they differentiate only into B lineage cells.

[0052] As used herein, the term "B-lineage cells" refers to a type of lymphocyte of the hematopoietic lineage that can differentiate from HSPCs, whether tissue-derived or PSC-derived, and that are more specialized / committed than B-cell precursors. More specifically, B-lineage cells can be derived from multilymphoid progenitors (MLPs) or common lymphoid progenitors (CLPs). Early B-lineage cells may be characterized by the expression of both CD10 and CD19 surface markers, while more mature naive B cells express CD19 but not CD10. In some embodiments, B-lineage cells may express CD19 (and not CD10) but still be distinguishable from B-cell precursors based on one or more other markers. In one embodiment, double-positive CD10 + CD19 +B lineage cells may lose expression of one or the other marker, but still remain committed to the B lineage. In one embodiment, after inducing B cell precursors according to the present disclosure, some CD19 + Cells may contain CD19 + The cells may be B-lineage cells or a subpopulation of more primitive cells. However, when these cells obtained after exposure to induction medium are exposed to differentiation medium, the proportion of B-lineage cells increases. In other words, the B-lineage cell population can be characterized by a higher proportion of cells expressing CD19 compared to the B-cell precursor population.

[0053] As used herein, the terms "B cell" or "B cells" refer to a cell type that differentiates from B-lineage cells. B cells are typically characterized by the absence of T-, NK-, and erythromyeloid-specific markers; expression of one or more of CD19, CD20, B cell receptor (i.e., surface IgM), IgG, IgD, IgA, IgE, and CD138; and their effector functions. More specifically, B cell effector functions can include antibody production. B cell types, plasma cells and plasmablasts, secrete antibodies and are characterized by expression of CD138 (plasmocytes), CD38, and CD27. B cell differentiation from PSCs or HSPCs is usually mediated by one or more progenitor cell populations, such as PSC-derived mesodermal precursors and / or lymphoid progenitors (e.g., PSC-derived lymphoid progenitors).

[0054] Media and Methods The disclosed methods involve differentiating hematopoietic progenitor cells derived from either tissue or PSCs into immature or mature B cells via one or more intermediate cell populations. The methods disclosed herein for differentiating HSPCs and other derived downstream cell types are preferably in vitro methods. The media disclosed below can be used to carry out the disclosed methods and are therefore independent aspects of the disclosure.

[0055] The directed differentiation method of the present disclosure involves the use of CD34 + This can include contacting the population of HSPCs with an induction medium and culturing the population of HSPCs in the induction medium for a time sufficient to induce a population of B cell precursors. In some embodiments, limited differentiation into B lineage cells can also occur during this stage.

[0056] In one embodiment, the directed differentiation method of the present disclosure induces a population of B cell precursors. In one embodiment, the directed differentiation method of the present disclosure induces a population of B lineage cells. In one embodiment, the directed differentiation method of the present disclosure induces an IgM + and / or induce a population of antibody-secreting cells.

[0057] The induction medium is any medium that can be used to differentiate HSPCs into a B cell precursor population. In a preferred embodiment, the induction medium is serum-free. When the induction medium is serum-free, it may be necessary to include a serum replacement supplement, such as BIT 9500 Serum Substitute (STEMCELL Technologies, model number 09500) or other commercially available serum replacement solutions. Alternatively, components normally present in serum that are necessary for culturing or differentiating any of the cells disclosed herein may be added individually to the induction medium at acceptable concentrations. In one embodiment, the component normally present in serum is albumin. When albumin is included in the induction medium (in place of serum), it can be from any species, but is typically either bovine or human. In some embodiments, the albumin can be recombinant.

[0058] The induction medium of the present disclosure, alternatively referred to as "SUPPLCTL" medium, includes a basal medium appropriately formulated to culture HSPCs and support the induction of B cell precursors. Accordingly, a suitable basal medium is suitable for culturing cells of the hematopoietic lineage, particularly B cell precursors and / or B lineage cells and / or IgM + Any basal medium that supports cell culture. Exemplary basal media include, but are not limited to, STEMdiff™ Hematopoietic-EB Basal Medium (STEMCELL Technologies, Model No. 100-171), STEMdiff™ Hematopoietic Basal Medium (STEMCELL Technologies, Model No. 05311), STEMdiff™ APEL™ 2 Medium (STEMCELL Technologies, Model No. 05270), StemSpan™ AOF Medium (STEMCELL Technologies, Model No. 100-0130), StemSpan™ SFEM & SFEM II, ImmunoCult™ XF (STEMCELL Technologies, Model Nos. 09650, 09655, 10981), or any other commercially available basal medium suitable for the purpose. Typical components for formulating a basal medium may include salts, buffers, lipids, amino acids, trace elements, specific proteins, vitamins, minerals, reducing agents, etc. In one embodiment, the basal medium is optimized to support the differentiation of HSPCs and the derivation of B cell precursor(s) therefrom.

[0059] In one embodiment, the induction medium comprises at least one of stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L). In one embodiment, the induction medium comprises two or more of SCF, TPO, and FLT3L. In one embodiment, the induction medium comprises each of SCF, TPO, and FLT3L. In one embodiment, the induction medium comprises either SCF or TPO. In one embodiment, the induction medium does not comprise one, two, or each of TPO, SCF, and FLT3L. In one embodiment, the induction medium does not comprise one or both of TPO and SCF. In one embodiment, the induction medium does not comprise TPO. In one embodiment, the induction medium does not comprise SCF.

[0060] When SCF is included in the induction medium, the concentration of SCF therein can be in the range of about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0061] When FLT3L is included in the induction medium, the concentration of FLT3L therein can be in the range of about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0062] When TPO is included in the induction medium, the concentration of TPO therein can be in the range of about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0063] In one embodiment, the induction medium further comprises at least one other cytokine. In one embodiment, the at least one other cytokine is one or more interleukins. In one embodiment, the at least one other cytokine is one or more of IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-15, IL-17, and IL-21. In one embodiment, the at least one other cytokine is IL-3, IL-6, or IL-7, or any combination thereof.

[0064] The concentration of at least one other cytokine included in the induction medium can be in the range of about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0065] In one embodiment, the induction medium further comprises one or more additional cytokines or growth factors, or small molecules to further enhance the induction of a B cell precursor population from the HSPC population. Non-limiting examples of the one or more additional cytokines or growth factors that may be included in the induction medium include erythropoietin (EPO), insulin growth factor 1 (IGF-1) and insulin growth factor 2 (IGF-2), B cell activating factor (BAFF), proliferation-inducing ligand (APRIL), and interferon gamma (IFN-g).

[0066] The concentration of one or more additional growth factors included in the induction medium can range from about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0067] In one embodiment, the induction medium can be formulated as a complete medium. In one embodiment, the induction medium can be freshly prepared before use, and thus the basal medium can be stored separately from one or more supplements added to the basal medium. In one example, growth factors and cytokines can be combined with one or more supplements and added to the basal medium just before using the complete induction medium in the induction / differentiation method.

[0068] The growth factors and cytokines included in the induction medium can be obtained from a variety of commercial suppliers and may also be recombinant.

[0069] The induction medium of the present disclosure can exhibit synergistic effects with substrates to support the culture of HSPC populations. In one embodiment, stromal cells or feeder cells can be used with the cell culture medium of the present disclosure. Non-exhaustive examples of such cells include embryonic liver cell line EL08.1D2, AFT024 cells, OP9 cells, MS-5 or M2-10B4 cells, mouse embryonic fibroblasts or stromal cells derived from embryonic aorta-gonad mesonephros (AGM).

[0070] In one embodiment, the HSPC population is cultured under feeder cell-free and / or stromal cell-free conditions. Such techniques may utilize medium pre-conditioned with stromal / feeder cells, or such systems may utilize stromal / feeder cell substitutes. The stromal / feeder cell substitutes may contain one or more defined components that provide the appropriate signals or contact sites to the cells in culture. Such components may be included (e.g., solubilized) in the induction medium or used as coatings applied to the interior culture surface of the culture vessel or on solid surfaces suspended in cell culture medium, e.g., particles, beads, microcarriers, etc. Non-exhaustive examples of such components may include fibronectin coatings, gelatin coatings, collagen coatings, immobilized Notch ligands, or coatings such as StemSpan™ Lymphoid Differentiation Coating Supplement (STEMCELL Technologies, Cat. No. 09925) or Matrigel (Corning).

[0071] In one embodiment, the HSPC population is cultured in the presence of an extracellular matrix protein or cell adhesion molecule (and in the absence of feeder cells and / or stromal cell carriers). In one embodiment, the extracellular matrix protein or cell adhesion molecule is solubilized in the induction medium or coated on a surface in contact with the induction medium. In one embodiment, the extracellular matrix protein is fibronectin, vitronectin, laminin, ECM1, SPARC, or osteopontin. In one embodiment, the cell adhesion molecule is a vascular cell adhesion molecule (e.g., VCAM-1) or an immobilized SCF protein (e.g., SCF-Fc).

[0072] In one embodiment, different combinations of the aforementioned proteins may be used. In one embodiment, a combination of extracellular matrix proteins is used. In one embodiment, a combination of cell adhesion molecules is used. In one embodiment, a combination of extracellular matrix protein(s) and cell adhesion molecule(s) is used.

[0073] When used, concentrations of extracellular matrix proteins or cell adhesion molecules that exhibit synergistic activity with induction medium range from about 0.1-100 μg / mL (or 0.03-30 μg / well in a 96-well plate), about 0.2-50 μg / mL (or 0.06-15 μg / well in a 96-well plate), or about 0.5-20 μg / mL (or 0.15-6 μg / well in a 96-well plate).

[0074] In one embodiment, the media of the present disclosure does not rely on the use in conjunction with extracellular matrix proteins or cell adhesion molecules.

[0075] Culturing the population of HSPCs in the induction medium can be for any period of time that does not affect their viability or ability to differentiate into downstream lineages. In one embodiment, the directed differentiation method for deriving a population of B cell precursors from a population of HSPCs involves the use of CD34 in the induction medium. + In one embodiment, the directed differentiation method for deriving a population of B cell precursors from a population of HSPCs comprises culturing the population of HSPCs for about 1 to 28 days, about 3 to 25 days, about 5 to 21 days, or about 7 to 14 days. + It involves culturing a population of HSPCs for approximately 3-14 days.

[0076] In one embodiment, 1% or more of the induced cells are B cell precursors (e.g., express CD10). In one embodiment, 5% or more of the induced cells are B cell precursors. In one embodiment, 10% or more of the induced cells are B cell precursors. In one embodiment, 20% or more of the induced cells are B cell precursors. In one embodiment, 30% or more of the induced cells are B cell precursors. In one embodiment, 40% or more of the induced cells are B cell precursors. In one embodiment, 50% or more of the induced cells are B cell precursors. Further, in one embodiment, 1% or more of the cells induced using the induction medium express CD19. In one embodiment, 5% or more of the cells induced using the induction medium express CD19. In one embodiment, 10% or more of the cells induced using the induction medium express CD19. In one embodiment, 20% or more of the cells induced using the induction medium express CD19. In one embodiment, 30% or more of the cells induced using the induction medium express CD19. In one embodiment, 40% or more of the cells induced using the induction medium express CD19.

[0077] In one embodiment, CD19, which may appear upon induction (using induction medium) of B cell precursors. + The cells can be B-lineage cells. In one embodiment, CD19 cells can appear upon induction (using induction medium) of B-cell precursors. + The cells may not be B-lineage cells, but may be more primitive cells or progenitor cells thereof. In one embodiment, both of the aforementioned cell types may be included in the cells obtained after culturing in induction medium.

[0078] In one embodiment, induction of B cell precursors using induction medium comprises one or more CD10 + Cells, ≥5 CD10 per input cell + Cells, ≥10 CD10 per input cell + Cells, ≥20 CD10 per input cell + Cells, ≥50 CD10 per input cell + ≥100 CD10 per cell or input cell +Furthermore, in one embodiment, induction of B cell precursors using induction medium results in one or more CD19 cells per input cell. + Cells, ≥5 CD19 per input cell + Cells, ≥10 CD19 per input cell + Cells, ≥20 CD19 per input cell + Cells, 50 or more CD19 per input cell + ≥100 CD19 per cell or input cell + Bring the cells.

[0079] In another aspect, the directed differentiation method of the present disclosure further comprises contacting the population of B cell precursors with a differentiation medium and culturing the population of B cell precursors in the differentiation medium for a time sufficient to obtain B lineage cells.

[0080] In one embodiment, the population of B cell precursors expresses one or both of CD10 or CD19.

[0081] In one embodiment, the B lineage cells are CD19 + In one embodiment, the B lineage cells are double positive CD10 + CD19 + In one embodiment, the population of B lineage cells is CD34 in the induction medium. + After culturing the HSPC population (i.e., after induction of the B cell precursor population using induction medium), more CD19 + In one embodiment, the population of B lineage cells contains more than twice as many CD19 cells as the population of B cell precursors after derivation. + In one embodiment, the population of B lineage cells contains three times more CD19 cells than after derivation of the population of B cell precursors. + In one embodiment, the population of B lineage cells is four times more CD19 positive than the population of B cell precursors after derivation. + In one embodiment, the population of B lineage cells contains 5 times more CD19 cells than after derivation of the population of B cell precursors. +In one embodiment, the population of B lineage cells contains 10 times more CD19 cells than after derivation of the population of B cell precursors. + In one embodiment, the population of B lineage cells contains 20 times more CD19 cells than after derivation of the population of B cell precursors. + In one embodiment, the population of B lineage cells contains 50 times more CD19 cells than after derivation of the population of B cell precursors. + Contains cells.

[0082] A differentiation medium is any medium that can be used to differentiate a B cell precursor population (to a B-lineage cell population). In a preferred embodiment, the differentiation medium is serum-free. When a differentiation medium is serum-free, it may be necessary to include a serum replacement supplement, such as BIT 9500 Serum Substitute (STEMCELL Technologies, model number 09500) or other commercially available serum replacement solutions. Alternatively, components normally present in serum that are necessary for culturing or differentiating any of the cells disclosed herein may be added individually to the differentiation medium at acceptable concentrations. In one embodiment, the component normally present in serum is albumin. When albumin is included in the differentiation medium (in place of serum), it can be from any species, but is typically either bovine or human. In some embodiments, the albumin can be recombinant.

[0083] The differentiation medium of the present disclosure, alternatively referred to as "DiM" medium, may comprise HSPCs and / or B cell precursors and / or B lineage cells and / or IgM +Basal media suitable for culturing cells include those formulated appropriately. Thus, a suitable basal medium is any basal medium that supports the culture of hematopoietic lineage cells, particularly B-lineage cells. Exemplary basal media include, but are not limited to, STEMdiff™ Hematopoietic-EB Basal Medium (STEMCELL Technologies, Model No. 100-171), STEMdiff™ Hematopoietic Basal Medium (STEMCELL Technologies, Model No. 05311), STEMdiff™ APEL™ 2 Medium (STEMCELL Technologies, Model No. 05270), StemSpan™ AOF Medium (STEMCELL Technologies, Model No. 100-0130), StemSpan™ SFEM & SFEM II, ImmunoCult™ XF (STEMCELL Technologies, Model Nos. 09650, 09655, 10981), or any other commercially available basal medium suitable for the purpose. Common components used to formulate such basal media may include salts, buffers, lipids, amino acids, trace elements, specific proteins, vitamins, minerals, reducing agents, etc. In one embodiment, the basal medium is optimized to support the differentiation of HSPCs and the derivation of B cell precursor(s) therefrom, and the further differentiation of B lineage cells.

[0084] In one embodiment, the differentiation medium comprises at least one of FLT3L, TPO, and SCF. In one embodiment, the differentiation medium comprises two or more of FLT3L, TPO, and SCF. In one embodiment, the differentiation medium comprises each of FLT3L, TPO, and SCF. In one embodiment, the differentiation medium comprises each of FLT3L, TPO, and SCF, and at least one other cytokine. In one embodiment, the differentiation medium comprises either SCF or TPO. In one embodiment, the differentiation medium does not comprise one or each of TPO, SCF, and FLT3L. In one embodiment, the differentiation medium does not comprise one or both of TPO and SCF. In one embodiment, the differentiation medium does not comprise TPO. In one embodiment, the differentiation medium does not comprise SCF.

[0085] When SCF is included in the differentiation medium, the concentration of SCF therein can be in the range of about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0086] When FLT3L is included in the differentiation medium, the concentration of FLT3L therein can be in the range of about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0087] When TPO is included in the differentiation medium, the concentration of TPO therein can be in the range of about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0088] In one embodiment, the differentiation medium further comprises at least one other cytokine. In one embodiment, the at least one other cytokine is one or more interleukins. In one embodiment, the at least one other cytokine is one or more of IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-15, IL-17, and IL-21. In one embodiment, the at least one other cytokine is IL-3, IL-6, or IL-7, or any combination thereof.

[0089] The concentration of at least one other cytokine in the differentiation medium can be in the range of about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0090] In one embodiment, the differentiation medium further comprises one or more additional cytokines or growth factors, or small molecules to further enhance the differentiation of B lineage cells from the B cell precursor population. Non-limiting examples of the one or more additional cytokines or growth factors that may be included in the differentiation medium include erythropoietin (EPO), insulin growth factor 1 (IGF-1) and insulin growth factor 2 (IGF-2), B cell activating factor (BAFF), proliferation-inducing ligand (APRIL), and interferon gamma (IFN-g).

[0091] The concentration of one or more additional cytokines or growth factors included in the differentiation medium can be in the range of about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0092] In one embodiment, the differentiation medium can be formulated as a complete medium. In one embodiment, the differentiation medium can be freshly prepared before use, and thus the basal medium can be stored separately from one or more supplements added to the basal medium. In one example, growth factors and cytokines can be combined with supplements and added to the basal medium just before using the complete differentiation medium in the induction / differentiation method.

[0093] The growth factors and cytokines included in the differentiation medium can be obtained from a variety of commercial suppliers and may also be recombinant.

[0094] The differentiation medium of the present disclosure may synergize with a substrate to support the culture of a population of B cell precursors (for differentiation of B lineage cells). In one embodiment, stromal cells or feeder cells may be used with the cell culture medium of the present disclosure. Non-exhaustive examples of such cells include embryonic liver cell line EL08.1D2, AFT024 cells, OP9 cells, MS-5 or M2-10B4 cells, mouse embryonic fibroblasts or stromal cells derived from the embryonic aorta-gonad mesonephros.

[0095] In one embodiment, the culture of the B cell precursor population is performed under feeder cell-free and / or stromal cell-free conditions. Such techniques may utilize medium pre-conditioned with stromal / feeder cells, or such systems may utilize stromal / feeder cell substitutes. The stromal / feeder cell substitutes may contain one or more defined components that provide the appropriate signals or binding sites to the cells in culture. Such components may be included in the differentiation medium or used as coatings applied to the interior culture surface of the culture vessel or on solid surfaces suspended in the cell culture medium, such as particles, beads, microcarriers, etc. Non-exhaustive examples of such components may include a fibronectin coating, a gelatin coating, a collagen coating, or Matrigel (Corning).

[0096] In one embodiment, the population of B cell precursors is cultured in the presence of an extracellular matrix protein or cell adhesion molecule (and in the absence of feeder cells and / or stromal cell carriers). In one embodiment, the extracellular matrix protein or cell adhesion molecule is solubilized in the differentiation medium or coated on a surface in contact with the differentiation medium. In one embodiment, the extracellular matrix protein is fibronectin, vitronectin, laminin, ECM1, SPARC, or osteopontin. In one embodiment, the cell adhesion molecule is a vascular cell adhesion molecule (e.g., VCAM-1) or an immobilized SCF protein (e.g., SCF-Fc).

[0097] In one embodiment, different combinations of the aforementioned proteins may be used. In one embodiment, a combination of extracellular matrix proteins is used. In one embodiment, a combination of cell adhesion molecules is used. In one embodiment, a combination of extracellular matrix protein(s) and cell adhesion molecule(s) is used.

[0098] If included, concentrations of extracellular matrix proteins or cell adhesion molecules that exhibit synergy with differentiation medium range from about 0.1-100 μg / mL (or 0.03-30 μg / well in a 96-well plate), about 0.2-50 μg / mL (or 0.06-15 μg / well in a 96-well plate), or about 0.5-20 μg / mL (or 0.15-6 μg / well in a 96-well plate).

[0099] In one embodiment, the media of the present disclosure does not rely on the use in conjunction with extracellular matrix proteins or cell adhesion molecules.

[0100] Culturing the population of B cell precursors in differentiation medium can be for any period of time that does not affect their viability or ability to differentiate into downstream lineages. In one embodiment, a method for differentiating a population of B cell precursors into a population of B lineage cells comprises culturing the population of B cell precursors in differentiation medium for about 1 to 28 days, about 3 to 25 days, about 5 to 21 days, or about 7 to 14 days. In one embodiment, a method for differentiating a population of B cell precursors into a population of B lineage cells comprises culturing the population of B cell precursors in differentiation medium for about 3 to 14 days.

[0101] In one embodiment, 1% or more of the cells differentiated using the differentiation medium are B lineage cells (e.g., express CD19). In one embodiment, 5% or more of the cells differentiated using the differentiation medium are B lineage cells. In one embodiment, 10% or more of the cells differentiated using the differentiation medium are B lineage cells. In one embodiment, 20% or more of the cells differentiated using the differentiation medium are B lineage cells. In one embodiment, 30% or more of the cells differentiated using the differentiation medium are B lineage cells. In one embodiment, 40% or more of the cells differentiated using the differentiation medium are B lineage cells. In one embodiment, 50% or more of the cells differentiated using the differentiation medium are B lineage cells. In one embodiment, 60% or more of the cells differentiated using the differentiation medium are B lineage cells. In one embodiment, 70% or more of the cells differentiated using the differentiation medium are B lineage cells. In one embodiment, 80% or more of the cells differentiated using the differentiation medium are B lineage cells. In one embodiment, 90% or more of the cells differentiated using the differentiation medium are B lineage cells.

[0102] In one embodiment, CD19 + At least some of the B lineage cells (those differentiated using differentiation medium) express IgM + In one embodiment, the cells are about 1% or more CD19 + B lineage cells express IgM + In one embodiment, the CD19 + B lineage cells express IgM +In one embodiment, the CD19 + B lineage cells express IgM + In one embodiment, the cells are about 4% or more CD19 + B lineage cells express IgM + In one embodiment, the cells are about 5% or more CD19 + B lineage cells express IgM + In one embodiment, the cells are about 10% or more CD19 + B lineage cells express IgM + It is a cell.

[0103] In one embodiment, differentiation of B lineage cells using a differentiation medium results in a cell population of 10 or more CD19 cells per input cell. + Cells, ≥25 CD19 per input cell + Cells, 50 or more CD19 per input cell + Cells, ≥100 CD19 per input cell + Cells, ≥250 CD19 per input cell + Cells, ≥500 CD19 per input cell + Cells, ≥1000 CD19 per input cell + ≥2000 CD19 per cell or input cell + Furthermore, in one embodiment, differentiation of B lineage cells using a differentiation medium results in one or more IgM cells per input cell. + Cells, 5 or more IgM per input cell + Cells, 10 or more IgM per input cell + Cells, 20 or more IgM per input cell + Cells, 50 or more IgM per input cell + 100 or more IgM per cell or input cells + Bring the cells.

[0104] In another aspect, the directed differentiation method of the present disclosure comprises contacting a population of B lineage cells with a downstream differentiation medium and +The method comprises culturing the population of B-lineage cells in downstream differentiation medium for a sufficient time to obtain and / or expand the cells (and / or antibody-secreting cells).

[0105] In one embodiment, the population of B lineage cells is double positive CD10 + CD19 + B lineage cells or single positive CD19 + CD19 on cells + It comprises or is a population of cells.

[0106] In one embodiment, the method involves the detection of CD19 in cells (e.g., a population of B-lineage cells) produced after culture in a differentiation medium of the present disclosure. + Cells (e.g., double-positive CD10 + CD19 + IgM after culture in downstream differentiation medium is more abundant than that contained in the population of B lineage cells. + Further comprising obtaining the cells.

[0107] Downstream differentiation medium (IgM + (which can be considered a medium for differentiation and expansion of cells) to differentiate a population of B lineage cells (IgM + to differentiate into a population of cells, or to IgM +The downstream differentiation medium is any medium that can be used to expand cells. In a preferred embodiment, the downstream differentiation medium is serum-free. When the downstream differentiation medium is serum-free, it may be necessary to include a serum replacement supplement, such as BIT 9500 Serum Substitute (STEMCELL Technologies, model number 09500) or other commercially available serum replacement solutions. Alternatively, components normally present in serum that are necessary for culturing or differentiating any of the cells of the present disclosure may be added individually to the downstream differentiation medium at acceptable concentrations. In one embodiment, the component normally present in serum is albumin. When albumin is included in the downstream differentiation medium (in place of serum), it can be derived from any species, but is typically either bovine or human. In some embodiments, the albumin can be recombinant.

[0108] The downstream differentiation medium of the present disclosure, alternatively referred to as "DDM" medium, is used to culture HSPCs and induce B cell precursors, IgM +Basal media suitable for supporting the differentiation of hematopoietic lineage cells, including B-lineage cells, include basal media formulated appropriately for supporting the differentiation of hematopoietic lineage cells, particularly B-lineage cells. Accordingly, suitable basal media are any basal media that support the culture of hematopoietic lineage cells, particularly B-lineage cells. Exemplary basal media include, but are not limited to, STEMdiff™ Hematopoietic-EB Basal Medium (STEMCELL Technologies, Model No. 100-171), STEMdiff™ Hematopoietic Basal Medium (STEMCELL Technologies, Model No. 05311), STEMdiff™ APEL™ 2 Medium (STEMCELL Technologies, Model No. 05270), StemSpan™ AOF Medium (STEMCELL Technologies, Model No. 100-0130), StemSpan™ SFEM & SFEM II, ImmunoCult™ XF (STEMCELL Technologies, Model Nos. 09650, 09655, 10981), or any other commercially available basal medium suitable for the purpose. Common components used to formulate such a basal medium may include salts, buffers, lipids, amino acids, trace elements, specific proteins, vitamins, minerals, reducing agents, etc. In one embodiment, the basal medium induces HSPCs to differentiate into B lineage cells (from which IgM + The cells are formulated to optimally support differentiation into stem cells (including stem cells).

[0109] In one embodiment, the downstream differentiation medium comprises a ligand for human CD40. The ligand for human CD40 can be isolated and / or used in its natural form. Alternatively, the ligand for human CD40 can be engineered for increased activity and / or half-life and / or stability. The ligand for CD40 can be natural or engineered, available from commercial sources, or recombinant.

[0110] If included, the ligand for CD40 in downstream differentiation may be present at a concentration ranging from about 10 ng / mL to 5 μg / mL, about 25 ng / mL to 2 μg / mL, about 50 ng / mL to 1 μg / mL, or about 100 ng / mL to 500 ng / mL.

[0111] In one embodiment, the downstream differentiation medium further comprises at least one other cytokine. In one embodiment, the at least one other cytokine is one or more interleukins. In one embodiment, the at least one other cytokine is one or more of IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-15, IL-17, and IL-21. In one embodiment, the at least one other cytokine is one or more of IL-2, IL-4, IL-6, IL-7, IL-10, or IL-21, or any combination thereof.

[0112] The concentration of the at least one other cytokine (or each of the at least one other cytokine) in the downstream differentiation medium can be in the range of about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0113] In one embodiment, the downstream differentiation medium comprises IgM from a B lineage cell population. + To further enhance differentiation of cells and antibody-secreting cells, one or more additional cytokines or growth factors, or small molecules may be included in the differentiation medium. Non-limiting examples of one or more additional cytokines or growth factors that may be included in the differentiation medium include erythropoietin (EPO), insulin growth factor 1 (IGF-1), insulin growth factor 2 (IGF-2), B-cell activating factor (BAFF), proliferation-inducing ligand (APRIL), and interferon gamma (IFN-g).

[0114] The concentration of one or more additional growth factors included in the downstream differentiation medium can be in the range of about 0.1 ng / mL to 1 μg / mL, about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0115] In one embodiment, the one or more additional growth factors included in the downstream differentiation medium can be selected from one or more of SCF, TPO, and FLT3L. In one embodiment, the downstream differentiation medium can include two or more of SCF, TPO, and FLT3L. In one embodiment, the downstream differentiation medium may include each of SCF, TPO, and FLT3L, or none of them. In one embodiment, the downstream differentiation medium does not include one or both of SCF and FLT3L. In one embodiment, the downstream differentiation medium does not include TPO and does not include one or both of SCF and FLT3L.

[0116] When SCF is included in the downstream differentiation medium, the concentration of SCF therein can be in the range of about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0117] When FLT3L is included in the downstream differentiation medium, the concentration of FLT3L therein can be in the range of about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0118] When TPO is included in the downstream differentiation medium, the concentration of TPO therein can be in the range of about 0.5 ng / mL to 500 ng / mL, about 1 ng / mL to 250 ng / mL, about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0119] In one embodiment, the downstream differentiation medium comprises a basal medium and one or both of a CD40 ligand and at least one other cytokine. In one embodiment, the downstream differentiation medium comprises a basal medium and one or more of a CD40 ligand, at least one other cytokine, and at least one additional cytokine. In one embodiment, the downstream differentiation medium comprises a basal medium and one or both of a CD40 ligand and at least one additional cytokine.

[0120] In one embodiment, the downstream differentiation medium can be formulated as a complete medium. In one embodiment, the downstream differentiation medium can be freshly prepared before use, and thus the basal medium can be stored separately from one or more supplements added to the basal medium. In one example, growth factors and cytokines can be combined with supplements and added to the basal medium just before using the complete downstream differentiation medium in the induction / differentiation method.

[0121] The growth factors and cytokines included in the downstream differentiation medium can be obtained from a variety of commercial suppliers and may also be recombinant.

[0122] The downstream differentiation medium of the present disclosure may exhibit synergistic effects with substrates to support the cultivation of populations of B-lineage cells. In one embodiment, stromal cells or feeder cells may be used with the cell culture medium of the present disclosure. Non-exhaustive examples of such cells include embryonic liver cell line EL08.1D2, AFT024 cells, OP9 cells, MS-5 or M2-10B4 cells, mouse embryonic fibroblasts or stromal cells derived from embryonic aorta-gonad mesonephros (AGM).

[0123] In one embodiment, the culture of the population of B-lineage cells is performed under feeder cell-free and / or stromal cell-free conditions. Such procedures may utilize medium pre-conditioned with stromal / feeder cells, or such systems may utilize stromal / feeder cell substitutes. The stromal / feeder cell substitutes may contain one or more defined components that provide the appropriate signals or binding sites to the cells in culture. Such components may be included in downstream differentiation medium or used as coatings applied to the interior culture surface of the culture vessel or on solid surfaces suspended in the cell culture medium, e.g., particles, beads, microcarriers, etc. Non-exhaustive examples of such components may include a fibronectin coating, a gelatin coating, a collagen coating, or Matrigel (Corning).

[0124] In one embodiment, the population of B-lineage cells is cultured in the presence of an extracellular matrix protein or cell adhesion molecule (and in the absence of feeder cells and / or stromal cell carriers). In one embodiment, the extracellular matrix protein or cell adhesion molecule is solubilized in the downstream differentiation medium or coated on a surface in contact with the downstream differentiation medium. In one embodiment, the extracellular matrix protein is fibronectin, vitronectin, laminin, ECM1, SPARC, or osteopontin. In one embodiment, the cell adhesion molecule is a vascular cell adhesion molecule (e.g., VCAM-1) or an immobilized SCF protein (e.g., SCF-Fc).

[0125] In one embodiment, a combination of extracellular matrix proteins and cell adhesion molecules is used. In one embodiment, a combination of extracellular matrix proteins is used. In one embodiment, a combination of cell adhesion molecules is used. In one embodiment, a combination of extracellular matrix protein(s) and cell adhesion molecule(s) is used.

[0126] If included, concentrations of extracellular matrix proteins or cell adhesion molecules that exhibit synergy with downstream differentiation media range from about 0.1-100 μg / mL (or 0.03-30 μg / well in a 96-well plate), about 0.2-50 μg / mL (or 0.06-15 μg / well in a 96-well plate), or about 0.5-20 μg / mL (or 0.15-6 μg / well in a 96-well plate).

[0127] In one embodiment, the media of the present disclosure does not rely on the use in conjunction with extracellular matrix proteins or cell adhesion molecules.

[0128] Culturing the population of B lineage cells in downstream differentiation medium can be for any period of time that does not affect their viability or ability to differentiate into downstream lineages. In one embodiment, the population of B lineage cells is cultured in a downstream differentiation medium for IgM + The method for differentiating the population of B-lineage cells into a population of IgM cells and / or antibody-secreting cells comprises culturing the population of B-lineage cells in downstream differentiation medium for about 1 to 28 days, about 3 to 25 days, about 5 to 21 days, or about 7 to 14 days. In one embodiment, the population of B-lineage cells is differentiated into IgM + The method for differentiating the population of cells involves culturing the population of B lineage cells in downstream differentiation medium for about 3 to 21 days.

[0129] In one embodiment, after culturing B lineage cells in downstream differentiation medium, 10% or more of the cells are CD19 + In one embodiment, after culturing B lineage cells in downstream differentiation medium, 20% or more of the cells are CD19 + In one embodiment, after culturing B lineage cells in downstream differentiation medium, 30% or more of the cells are CD19 + In one embodiment, after culturing B lineage cells in downstream differentiation medium, 40% or more of the cells are CD19 + In one embodiment, after culturing B lineage cells in downstream differentiation medium, 50% or more of the cells are CD19 + In one embodiment, after culturing B lineage cells in downstream differentiation medium, 60% or more of the cells are CD19 +In one embodiment, after culturing B lineage cells in downstream differentiation medium, 70% or more of the cells are CD19 + In one embodiment, after culturing B lineage cells in downstream differentiation medium, 80% or more of the cells are CD19 + In one embodiment, after culturing B lineage cells in downstream differentiation medium, 90% or more of the cells are CD19 + It is a cell.

[0130] In one embodiment, CD19 after culturing B lineage cells in downstream differentiation medium + 1% of cells are IgM + In one embodiment, CD19 cells are obtained after culturing B lineage cells in downstream differentiation medium. + 2% of cells are IgM + In one embodiment, CD19 cells are obtained after culturing B lineage cells in downstream differentiation medium. + 3% of cells are IgM + In one embodiment, CD19 cells are obtained after culturing B lineage cells in downstream differentiation medium. + 4% of cells are IgM + In one embodiment, CD19 cells are obtained after culturing B lineage cells in downstream differentiation medium. + More than 5% of cells are IgM + In one embodiment, CD19 cells are obtained after culturing B lineage cells in downstream differentiation medium. + More than 10% of cells are IgM + In one embodiment, CD19 cells are obtained after culturing B lineage cells in downstream differentiation medium. + More than 15% of cells are IgM + In one embodiment, CD19 cells are obtained after culturing B lineage cells in downstream differentiation medium. + More than 20% of cells are IgM + It is a cell.

[0131] In one embodiment, downstream differentiation of B lineage cells using downstream differentiation medium is performed using a medium containing 50 or more CD19 + Cells, ≥100 CD19 per input cell + Cells, ≥250 CD19 per input cell+ Cells, ≥500 CD19 per input cell + Cells, ≥1000 CD19 per input cell + Cells, 2000+ CD19 per input cell + Cells, ≥3000 CD19 per input cell + 4000 or more CD19 per cell or input cells + Furthermore, in one embodiment, downstream differentiation of B lineage cells using downstream differentiation medium results in 10 or more IgM cells per input cell. + Cells, 25 or more IgM per input cell + Cells, 50 or more IgM per input cell + Cells, 100 or more IgM per input cell + Cells, 150 or more IgM per input cell + ≥200 IgM per cell or input cell + Bring the cells.

[0132] In one embodiment, after culture in downstream differentiation medium, about 0.5% or more of the produced cells are antibody-secreting cells. In one embodiment, about 1% or more of the produced cells are antibody-secreting cells. In one embodiment, about 2% or more of the produced cells are antibody-secreting cells. In one embodiment, about 3% or more of the produced cells are antibody-secreting cells. In one embodiment, about 4% or more of the produced cells are antibody-secreting cells. In one embodiment, about 5% or more of the produced cells are antibody-secreting cells. In one embodiment, about 10% or more of the produced cells are antibody-secreting cells.

[0133] In one embodiment, the antibody-secreting cells are CD19 + In one embodiment, the antibody-secreting cells are CD19- and such antibody-secreting cell populations are CD138 + In one embodiment, the antibody-secreting cells may be IgM - In one embodiment, the antibody-secreting cells secrete either IgM or IgG.

[0134] In embodiments of the methods disclosed herein in which the HSPCs are derived from PSCs, the PSCs may be cultured under serum-free conditions. In the same or different embodiments, the PSCs may be cultured under stromal cell-free and / or feeder-free conditions. CD34 from PSCs + Differentiation of HSPCs can be performed using commercially available kits such as the STEMdiff™ Hematopoietic Kit (STEMCELL Technologies) or STEMdiff™ Hematopoietic-EB Basal Medium in conjunction with EB Supplement A and EB Supplement B (STEMCELL Technologies). At this step, or any step of the methods disclosed herein, it may be desirable to purify / enrich the cells of interest before proceeding to the next step of the method. Methods for purifying / enriching cells are known, such as by immunomagnetic cell separation or fluorescence-activated cell sorting.

[0135] IgM + B cells are IgM + IgD + to naive B cells (IgM + IgD + through a transitional B cell stage) to IgM antibody-secreting cells, or IgG depending on isotype switching + , IgE + , or IgA + They can further mature into either memory B cells or plasma cells, which may also differentiate into antibody-secreting cells (capable of secreting IgM, IgG, IgE, or IgA).

[0136] In one embodiment, a method of deriving a population of B cell precursors from a population of PSC-derived HSPCs may comprise forming the PSCs into aggregates before differentiating the PSCs into HSPCs as described above, and then subjecting such PSC-derived HSPCs to induction medium conditions.

[0137] PSCs can be formed into aggregates using any known method. For example, PSC aggregates can be formed by depositing a desired number of PSCs on the bottom of a tube or well of a cell culture plate. Alternatively, aggregates can be formed by depositing a desired number of PSCs in the well of an Aggrewell™ microwell device (STEMCELL Technologies) to ensure efficient and reproducible formation of uniformly sized aggregates of PSCs.

[0138] In one embodiment, the number of PSCs used to form the aggregates is about 1 to 100,000. In one embodiment, the number of PSCs used to form the aggregates is about 10 to 10,000. In one embodiment, the number of PSCs used to form the aggregates is about 100 to 1,000.

[0139] Thus, in one embodiment, aggregates of PSCs can be formed in a microwell device. In one embodiment, aggregates of PSCs are formed from about 1000 cells or about 500 cells.

[0140] i) enriching HSPCs, ii) differentiating PSCs into mesodermal precursors, iii) deriving a population of B cell precursors from HSPCs, whether PSC- or tissue-derived, iv) differentiating B lineage cells from B cell precursors, and v) differentiating IgM from B lineage cells. + and / or differentiate IgM-secreting cells, or vi) differentiate IgG from B lineage cells or later cell types. + In addition to disclosing media and methods for differentiating PSCs and / or IgG-secreting cells, the various media disclosed herein can be included in systems or kits for the stepwise differentiation of PSCs or tissue-derived HSPCs into immature or mature B cells, whether or not under serum-free and / or feeder- or stromal-cell-free conditions.

[0141] In some embodiments, the entire system is performed under serum-free and / or feeder- or stromal-free conditions. In some embodiments, only certain aspects of the system are performed under serum-free and / or feeder- or stromal-free conditions. For example, without limiting the generality of the foregoing, differentiation of PSCs to mesodermal precursors, PSC-derived mesodermal precursors to hematopoietic progenitor cells, PSC-derived hematopoietic progenitor cells to lymphoid progenitor cells (e.g., a population of B cell precursors), differentiation of B cell precursors to B lineage cells, and differentiation of B lineage cells to either IgM-expressing cells (and / or IgM-secreting cells) and / or IgG-expressing cells (and / or IgG-secreting cells) is performed under serum-free and / or feeder- or stromal-free conditions, while further downstream steps may or may not be performed under these conditions. Conversely, the later stages of the system may be performed under serum-free and / or stroma-free conditions, while the earlier stages may or may not be performed under serum-free and / or feeder-cell-free or stroma-cell-free conditions.

[0142] In one embodiment, such a system or kit, in the context of a PSC workflow, includes a first culture system for differentiating PSCs into mesodermal progenitors; a second culture system for differentiating PSC-derived mesodermal progenitors into hematopoietic progenitor cells; a third culture system for differentiating PSC-derived hematopoietic progenitor cells into one or more subpopulations of lymphoid progenitor cells; a fourth culture system for differentiating PSC-derived lymphoid progenitor cells (e.g., a population of B cell precursors); a fifth culture system for differentiating PSC-derived lymphoid progenitor cells (e.g., a population of B cell precursors) into a population of B lineage cells; a sixth culture system for differentiating HSPCs into IgM-expressing cells (and / or IgM-secreting cells) and / or IgG-expressing cells (and / or IgG-secreting cells); a coated substrate; a first kit for positively or negatively enriching a population of HSPCs; a second kit for positively or negatively enriching a population of B cell precursors; a third kit for positively or negatively enriching a population of B lineage cells; a fourth kit for positively or negatively enriching a population of immature B cells; and a fifth kit for positively or negatively enriching a population of mature B cells.

[0143] In one embodiment, such a system or kit comprises a primary CD34 + Cell- or tissue-derived CD34 + In the context of cellular workflow, CD34 + The first culture system is for inducing B cell precursors from a population of HSPCs; the second culture system is for differentiating B lineage cells from a population of B cell precursors; and the second culture system is for differentiating IgM cells from a population of B lineage cells. +or a third culture system for further differentiating IgM-secreting cells; a coated substrate; a first kit for positively or negatively enriching a population of HSPCs; a second kit for positively or negatively enriching a population of B cell precursors; a third kit for positively or negatively enriching a population of B lineage cells; a fourth kit for positively or negatively enriching a population of immature B cells; and a fifth kit for positively or negatively enriching a population of mature B cells.

[0144] The cells obtained using the media disclosed herein or by the methods disclosed herein can be used for any downstream assay or purpose. In one embodiment, the cells of the present disclosure (whether a population of B cell precursors or CD19 + or CD10 + CD19 + B-lineage cell populations or IgM + These cells (whether IgM-secreting or IgM-secreting) can be used in research applications to investigate B cell development or the biology of B cell diseases such as cancer.

[0145] In one embodiment, the cells of the present disclosure may be used for transplantation purposes into a patient in need, such as a patient suffering from a hematopoietic (e.g., B cell) disorder. In such an embodiment, the cells transplanted into the patient in need comprise a population of B cell precursors, CD19 + or CD10 + CD19 + B lineage cell population, or IgM + The transplanted cells may be PSC-derived cells or cells secreting Ig, such as IgM and / or IgG. Such cells may be edited using known gene editing techniques to introduce one or more transgenes, remove one or more DNA fragments, or create one or more hypomorphic or hypermorphic mutations. In one embodiment, the transplanted cells are derived from PSCs, and therefore may be a universal source of allogeneic cells. In one embodiment, the transplanted cells are derived from tissues, and therefore may be a source of autologous cells.

[0146] In one embodiment, the cells of the present disclosure can be used to evaluate the responsiveness of cells to test conditions, such as toxicity studies or drug screening. In such embodiments, the source cells can correspond to a non-disease state or a disease state. In some embodiments, a disease state can be introduced into one or more starting cells, such as by gene editing techniques.

[0147] The following non-limiting examples illustrate the present disclosure. [Example]

[0148] Example 1: CD34 from donor samples + HSPC enrichment Umbilical cord blood units were obtained from a commercial supplier and CD34 positively selected using the EasySep Human Cord Blood CD34 Positive Selection Kit II (STEMCELL Technologies). + HSPCs were enriched and then frozen in serum containing 10% DMSO or used fresh. + The cells were stained and then - (Lin included CD3, CD14, CD15, CD16, CD19, CD56, and CD66b), CD34, called "pop1" + CD38 - / mid / low CD10 - Cells were sorted (FACS Aria) for 100% of the total population. A second, more definitive sorted population, used as a control, was a Lin - CD34 + CD38 mid CD10 + (Figure 1).

[0149] Example 2: Flow cytometry and determination of cell number and yield At any stage of the directed differentiation protocol outlined in this disclosure, samples can be collected and their phenotype assessed by flow cytometry. The following schematic protocol applies equally to measuring CD34, CD10, CD19, CD20, and IgM.

[0150] Briefly, cell samples were collected by centrifugation and washed appropriately. Subsequently, the cell samples were stained with fluorophore-conjugated antibodies against selected antigens and analyzed using a CytoFLEX S™ flow cytometer (Beckman-Coulter). Dead cells were excluded by light scattering profile and DRAQ7 staining.

[0151] Total viable cell counts were obtained using a NucleoCounter NC250 (Chemometec) according to the manufacturer's recommendations. After diluting cells as needed, they were stained with a mixture of acridine orange and DAPI (AO / DAPI). In this mixture, AO labels the cell membrane, and DAPI labels the nucleic acids of dead / dying cells, allowing for photographic discrimination of viable and nonviable cells in a sample. The NC250 software then analyzed the resulting images and recorded the cell count, including the viable cell concentration. To calculate the yield of a specific cell type per input cell, the total viable count was multiplied by the % abundance of a given cell type. For example, for input CD34 + CD10 per cell + To calculate cell yield, we first counted viable cells and then analyzed CD10 cells obtained by flow cytometry. + This number is then divided by the number of input cells (input CD34 + (For cells), the final value is obtained by dividing the CD34 + By multiplying the cell abundance ratio, the input CD34 + Cell counts were obtained.

[0152] Example 3: Cord blood-derived CD34 + Induction of B cell precursors from HSPCs Enriched CD34 of Example 1+ HSPCs were differentiated into B cell precursors in induction medium, which typically contained a basal medium such as StemSpan™ SFEMII (STEMCELL Technologies, model no. 09655) and various stage-specific cytokines and growth factors. The first replicate of induction medium contained SCF, TPO, FLT3L, and IL-7, and experiments testing various combinations of such factors were performed (Figures 2, 3, and 6).

[0153] Briefly, CD34 was isolated essentially as described in Example 1. + Pop2 cells were sorted from the enriched population of HSPCs and seeded at 5000 cells per well of a 24-well plate in the induction medium formulations tested. After 14 days of culture in the various medium formulations, CD10 + (Figure 2A) and CD19 + The produced cells were analyzed by flow cytometry for abundance and yield of cells (Figure 2B).

[0154] TPO is a cord blood-derived CD34 + It was observed that TPO was not essential in the induction medium for inducing B cell precursors from HSPCs. Having determined that TPO was not essential in the induction medium, the effects of other cytokines (e.g., IL-3 and / or IL-6) added to such induction medium were examined.

[0155] Briefly, pop2 cells were transfected with CD34 + HSPCs were sorted from the enriched population, seeded, and cultured essentially as described above. After 14 days of culture in various media formulations, the total fold expansion (Figure 3A), CD10 + Cell abundance and yield (Figure 3B), as well as CD19 + The produced cells were analyzed by flow cytometry for cell abundance and yield (Figure 3C). An exemplary plot of cells analyzed by flow cytometry at day 14 is shown in Figure 3D.

[0156] Inclusion of IL-3 or IL-6 alone in the induction medium, without combination, inhibited CD10 expression from pop2 cells. + The cell yield was improved, and IL-3 alone significantly increased the CD19 expression levels from pop2 cells. + Improved cell yield was observed.

[0157] Experiments similar to those performed and reported above were performed using pop1 cells enriched, sorted, and seeded as described in Example 1. After 14 days of culture in various media formulations, the total fold expansion (Fig. 3E), CD10 + Cell abundance and yield (Figure 3F), as well as CD19 + The produced cells were analyzed by flow cytometry for cell abundance and yield (Figure 3G). An exemplary plot of cells analyzed by flow cytometry at day 14 is shown in Figure 3H.

[0158] The inclusion of IL-3 or IL-6, alone or in combination, significantly reduced CD10 expression from pop1 cells after 14 days in induction medium. + An improved cell yield was observed.

[0159] Example 4: CD19 from cord blood-derived B cell precursors + B-lineage cell differentiation CD34 essentially as described in Example 3 + B cell precursors were derived from HSPCs (i.e., pop2 cells) and expressed CD19 as described below. + They were differentiated into B lineage cells.

[0160] In the first experiment, pop2 cells were cultured in control induction medium (e.g., SUPPLCTL) for 14 days. After 14 days, the cells were transferred to various differentiation medium formulations and cultured for an additional 14 days. The differentiation medium conditions tested were formulated to lack one or more of SCF, TPO, IL-7, or FLT3L, as shown in Figure 4. On day 28, cells were analyzed by flow cytometry for CD10 + cells (Figure 4A), CD19 + B-lineage cells (Figure 4B), and CD19+ Cell-dependent IgM + The abundance and yield of cells (Fig. 4C) were analyzed.

[0161] It was observed that all differentiation media formulations resulted in comparable abundance and yield in each cell population analyzed.

[0162] In follow-up experiments, B cell precursors were induced from pop2 cells for 14 days using induction medium as described in Example 3. After 14 days, producer cells were harvested, counted, and analyzed for CD10 and CD19 expression by flow cytometry, with 1-2 x 10 cells per well of a 24-well plate. 5 The cells were replated in differentiation medium as described in the paragraph above. After 2 weeks of culture, all cells were harvested, counted, and analyzed by flow cytometry for expression of CD10, CD19, and IgM (data not shown). The inclusion of IL-6 in the induction medium synergized with the differentiation medium, resulting in the highest number of CD19 cells. + It was observed that B lineage cells were obtained (data not shown).

[0163] To confirm the above findings, pop2 cells were cultured in induction medium formulation (as described in Example 3 and this Example) for 14 days, and then the resulting cells were harvested, counted, and analyzed by flow cytometry for the expression of markers such as CD10 and CD19. Day 14 cells were cultured in differentiation medium formulation (as described above in this Example) for an additional 14 days, then harvested, counted, and analyzed by flow cytometry for the expression (as well as the abundance and yield) of CD10 (Figure 5A), CD19 (Figure 5B), and IgM (Figure 5C). Pop1 cells were similarly cultured and analyzed by flow cytometry for the expression (as well as the abundance and yield) of CD10 (Figure 5D), CD19 (Figure 5E), and IgM (Figure 5F).

[0164] For pop2 cells, the developed induction and differentiation protocol resulted in approximately 150 CD19 cells per input cell. +Robust yields of B lineage cells could be generated. Furthermore, approximately 10 IgM per input cell + We were able to generate enriched CD34 cells. + Considering that only 5% of HSPCs represent pop2 cells, these efficiencies in serum-free and feeder-free conditions are remarkable. Although starting from pop1 cells, the developed induction and differentiation protocol resulted in the generation of approximately 300 CD19 per input cell, as observed in the context of pop2 cells. + Robust yields of B-lineage cells could be generated (Figure 5E). Furthermore, over 5 IgM per input cell + Cells were successfully generated (Figure 5F).

[0165] Similar to the experiments performed and reported above in this example, pop1 cells were enriched and isolated as described in Example 1 and then seeded at 5000 cells per well of a 24-well plate in an induction medium formulation lacking SCF but containing either IL-3 or IL-6, alone or in combination. After 14 days, producer cells were harvested, counted, and analyzed for CD10 and CD19 expression by flow cytometry, yielding 1-2 x 10 5 Cells / well were replated in differentiation medium (as described above in this example). After 14 days of culture, all cells were harvested, counted, and analyzed by flow cytometry for CD10, CD19, and IgM expression (data not shown). Cells on day 28 were harvested, counted, and analyzed by flow cytometry for CD10 (Figure 6A) and CD19 (Figure 6B) expression, as described above.

[0166] In this case, we started with pop1 cells, but as observed in the context of pop2 cells, the inclusion of IL-6 in the various induction medium formulations synergized with the differentiation medium, resulting in the highest abundance and yield of CD19. + B lineage cells were obtained.

[0167] CD19 + B lineage cells and IgM +To increase cell abundance and yield, the culture period in differentiation medium was extended from 14 to 28 days. After a 42-day protocol (14 days in induction medium and 28 days in differentiation medium), pop1-derived CD19 + B-lineage cell abundance and yield (Fig. 7A) and IgM among CD19+ cells + The abundance of CD34 cells (Figure 7B) was significantly increased. + Although pop1 cells are more abundant than pop2 cells in the enriched population of HSPCs, the results achieved using serum-free and feeder-free conditions are noteworthy.

[0168] Each starting cell population (bulk CD34 + , pop1 and pop2), CD19 + A general overview of B lineage cell abundance and yield is shown in Figure 8. Each cell population was separately subjected to induction conditions as described in Example 3 and this Example for 14 days, and then transferred to differentiation conditions as described in this Example for 14 days. + Regardless of the starting population of cells, it is clear that by using the present media and practicing the methods of the present disclosure, significant abundances and yields of CD19+ cells can be achieved.

[0169] Example 5: Cord blood-derived CD19 + B lineage cells to IgM + Optimization of downstream differentiation into cells By extending the culture period in differentiation medium, IgM + It was possible to improve the production of IgM cells. + Other methods to enhance cell production were explored. Specifically, IgM + The effects of culture medium additives to enhance cell yield were investigated.

[0170] Briefly, 5000 pop2 cells were seeded in each well of a 24-well plate in induction medium formulation (see Figure 3), and after 14 days of culture, producer cells were cultured at 1–2 × 10 cells per well of a 24-well plate. 5The cells were replated in differentiation medium (see Figures 4 and 5) at 100 cells / well for an additional 14 days. After 28 days of culture, the resulting cells were grown at 1–2 × 10 cells / well in a 24-well plate. 5 The cells were replated at 1000 x g and cultured for 7 days in various downstream differentiation media: basal SFEM II medium (STEMCELL Technologies) without cytokines or growth factors ("-DDM"), SFEM II (STEMCELL Technologies) basal medium supplemented with CD40L and a combination of cytokines ("DDMa"), and differentiation medium supplemented with CD40L and a combination of cytokines ("DDMb"). After the 35-day protocol, the produced cells were harvested, counted, and analyzed by flow cytometry for CD19 (Figure 9A) and IgM (Figure 9B). IgM and IgG secretion by the produced cells on day 35 was confirmed by ELISPOT (ImmunoSpot Human IgM / IgG-B-Cell Specific Double-Color ELISPOT kit). Compared to the StemSpan™ SFEMII basal medium control (Figure 9Ci), increased numbers of both IgM (red / gray dots) and IgG (blue / black dots) were detected in downstream differentiation medium supplemented with CD40L (Figures 9Cii and 9Ciii). Indeed, production (in terms of yield) of CD19- and IgM-expressing cells was achieved in CD40L-containing conditions.

[0171] For pop2 cells, starting from bulk cells, IgM + The effects of culture medium additives on cell differentiation were investigated. Bulk cells were seeded, cultured, and analyzed as described for pop2 cells (Figures 9D and 9E). As observed with pop2 cells, bulk cells in downstream differentiation medium formulations containing CD40L showed a significant increase (in terms of yield) in the production of CD19- and IgM-expressing cells compared to control conditions.

[0172] Example 6: Maintenance of PSCs When hPSCs were used to derive B cell precursors, they were maintained on Matrigel™-coated plates in mTeSR™1 (STEMCELL Technologies), TeSR™-E8 (STEMCELL Technologies), or mTeSR™ Plus (STEMCELL Technologies) medium for 6–8 days according to the manufacturer's recommendations. Complete medium changes were performed as needed. PSC colonies were passaged as clumps onto freshly coated Matrigel™ plates during maintenance culture. When PSCs were used for downstream assays, colonies were dissociated using ACCUTASE™ (STEMCELL Technologies) according to the manufacturer's recommended protocol to obtain single-cell suspensions.

[0173] Example 7: Formation and differentiation of PSC aggregates Prior to obtaining a single cell suspension according to Example 6, 24-well or 6-well Aggrewell™ 400 plates (STEMCELL Technologies) were prepared according to the manufacturer's recommendations, using anti-adhesion wash solution (STEMCELL Technologies) to reduce cell adhesion to the microwell device. After the recommended incubation, the anti-adhesion wash solution was discarded, and each well was rinsed once with an equal volume of DMEM-F12 containing 15 mM HEPES.

[0174] After preparing the microwell device, hPSCs dissociated according to Example 6 were seeded in one or more wells of the microwell device in EB Formation Medium (STEMdiff™ Hematopoietic-EB Basal Medium supplemented with STEMdiff™ Hematopoietic-EB Supplement A (STEMCELL Technologies)) and 10 μM Y-27632 (STEMCELL Technologies). In an embodiment using a 6-well format microwell device, 2.5 mL of EB Formation Medium was added to the wells of the microwell device. Next, an additional 2.5 mL of cell suspension (approximately 1.4 x 10 6 EB Formation Medium containing 1000 cells / mL was added to the wells, the microwell device was briefly centrifuged, and the device was incubated at 37°C. When using a 24-well microwell device, the volume per well must be reduced to 2 mL / well. The final cell concentration in each well of the microwell device was approximately 3 × 10 5 cells / ml or 6 × 10 in a 24-well plate 5 cells / well, or 7 x 10 5 cells / mL, or 3.5 x 10 in a 6-well plate 6 There should be 1 cell / well.

[0175] Example 8: Differentiation of aggregates into hematopoietic progenitor cells Aggregates were prepared as described in Example 7, and two days after aggregate formation, 2.5 mL of medium in each well of the microwell device was carefully removed and discarded without disrupting the aggregates. A 2.5 mL volume of fresh EB Medium A (STEMdiff™ Hematopoietic-EB Basal Medium (STEMCELL Technologies) supplemented with STEMdiff™ Hematopoietic-EB Supplement A (STEMCELL Technologies)) was added to each well, and the microwell device was incubated at 37°C.

[0176] On day 3, when mesodermal intermediates (e.g., mesodermal precursors) were formed, 2.5 mL of medium in each well of the microwell device was carefully removed and discarded without disrupting the aggregates. 2.5 mL of fresh EB Medium B (STEMdiff™ Hematopoietic-EB Basal Medium (STEMCELL Technologies)) supplemented with STEMdiff™ Hematopoietic-EB Supplement B (STEMCELL Technologies) was added to each well and incubated at 37°C to differentiate the mesodermal precursor cells into hematopoietic progenitor cells.

[0177] On day 5, aggregates were collected from each well of the microwell device and passed through a 37 μm reversible filter (STEMCELL Technologies) to isolate the aggregates on its surface. The aggregate filtrate was deposited into a fresh tube by inverting the filter over a fresh tube and drawing 2.5 mL / well of fresh EB Medium B against the mesh (1 mL / well was used for a 24-well microwell device). After gently resuspending the aggregates, the total volume was added to a non-tissue culture-treated plate and then incubated at 37°C. On day 7, 2.5 mL of fresh EB Medium B was added to each well of a 6-well plate (or 1 mL / well for a 24-well plate) and then incubated at 37°C. On day 10, half of the medium was replaced with fresh EB Medium B, taking care not to disrupt the aggregates, followed by an additional 2 days of incubation at 37°C.

[0178] Example 9: Enrichment of hematopoietic progenitor cells The aggregates from Example 8 were collected from each well and transferred to individual 15 mL tubes. The tubes were centrifuged at 300 × g for 5 to 10 minutes. The supernatant was aspirated, and 1 mL of Collagenase Type II-2500 U / mL (STEMCELL Technologies) (product number 07418) was added to each tube and incubated at 37°C for 20 minutes. 3 mL of TryPLE™ Express was then added, and each tube was incubated for an additional 20 minutes.

[0179] After incubation, 6 mL of DMEM / F12 was added to each tube and filtered through a 37 μm filter. The eluate was centrifuged at 300 × g for 5–10 minutes, and the supernatant was discarded. The pelleted cells were analyzed for CD34 + The cells were subjected to an enrichment protocol (EasySep™ Human CD34 Positive Selection Kit II, STEMCELL Technologies). + For enrichment, the manufacturer's recommendations were followed, except that the number of magnetic separation rounds was reduced from 4 to 2. H1, H9, WLS-1C, STiPS-M001, and STiPS-F016 PSC lines were CD34 + They differentiated efficiently into hematopoietic progenitor cells.

[0180] Example 10: hPSC-derived CD34 + Induction of B cell precursors from HSPCs Enriched CD34 of Example 9 + HSPCs were cultured at 2.5 x 10 cells per well of a 24-well plate. 4H9-derived cells were seeded at a density of 100 μg / ml and cultured for 14 days in induction medium in the presence of various coatings of extracellular matrix proteins or cell adhesion molecules or MS-5 stromal cells. The induction medium typically contained a basal medium such as StemSpan™ SFEM II (STEMCELL Technologies) and various stage-specific cytokines and growth factors, as in Examples 3 and 4. The first replicate of induction medium was formulated as above, but with the addition of IGF-1, and the resulting H9-derived cells after 14 days were analyzed for CD10 and CD19 expression.

[0181] The highest abundance of B cell precursors was generated when hPSCs were cultured in wells coated with either fibronectin or collagen 1, which performed better than more conventional approaches such as Matrigel™ coating (Figure 10A) or on MS-5 stromal cells (data not shown). We also observed that a significant abundance of B cell precursors was generated when hPSCs were cultured in wells that were not coated with extracellular matrix or that were coated with VCAM-1 and / or SCF-Fc (Figure 10B).

[0182] PSC-derived CD34 differentiates into B-lineage cells + Regarding the potential of HSPC cells, the expression of marker transcription factors in such cells was examined using cord blood-derived CD34 + The expression levels of PSC-derived CD34 were compared with those of HSPC cells. + HSPC and cord blood-derived CD34 + We observed a time-dependent decrease in the relative fold expression of the B cell specification transcription factor EBF1 in both HSPCs and PSCs (Figure 10C). The fold expression change of the B cell commitment factor PAX5 was significantly greater in PSC-derived CD34 cells than in HSPCs. + HSPC and cord blood-derived CD34 + Both PSC and HSPC cells increased during the differentiation protocol (Figure 10D), suggesting that PSC-derived differentiated cells have a similar transcriptional program to their cord blood-derived counterparts.

[0183] Example 11: CD19 from hPSC-derived B cell precursors + B-lineage cell differentiation B cell precursors generated as described in Example 10 were cultured at 5.0 x 10 cells per well of a 24-well plate in plates coated or uncoated with VCAM-1 and / or SCF-Fc. 4 The cells were replated onto VCAM-1-coated plates and cultured for 14 days in differentiation medium essentially as described in Example 4, but additionally containing IGF-1. H9-derived cells were harvested on day 28 and analyzed by flow cytometry for expression of CD10 and CD19 (Figure 10E) and CD19 and CD20 (Figure 10E).

[0184] The population of H9-derived cells expressing CD19 also expressed CD20, indicating that the serum-free and feeder-free conditions described were sufficient to induce CD19 expression. + This suggests that they may generate B-lineage cells.

[0185] Example 12: Cord blood-derived CD34 in the presence of a coating + B cell precursors from HSPCs and CD19 + Generation of B-lineage cells Induction of B cell precursors and CD19 from pop1 cells + The effect of extracellular matrix protein coating or cell adhesion molecule coating (vs. no coating) in the context of differentiation of B cell precursors into B lineage cells was also examined.

[0186] Briefly, pop1 cells were sorted as described in Example 1 and seeded at a density of 5000 cells per well of 24-well plates in TPO-free induction medium essentially as described in Example 3 (Figure 2). After 14 days of culture in induction medium in the presence of various coatings, each producer cell population was harvested and CD10 + (Figure 11A) and CD19 +The abundance and yield of the cells (Figure 11B) were analyzed by flow cytometry. Next, the cells on day 14 were plated into corresponding coated wells of a 24-well plate at 1 to 1.5 x 10 cells per well. 5 The cells were replated at 100x the number of cells per well and cultured in the presence of differentiation medium essentially as described in Example 4. Cells were harvested on day 28 and identified as CD19 + Cell abundance and yield (Fig. 11C) and CD19 + IgM in cells + The cell abundance (FIG. 11D) was analyzed by flow cytometry.

[0187] The coatings tested did not appear to significantly affect the induction of B cell precursors at day 14, nor did they positively affect the generation of CD19-expressing cells at day 14. Interestingly, certain of the coatings tested appeared to be potentially detrimental to the generation of CD19-expressing cells at day 14. + The same overall conclusions can be drawn for the generation of B lineage cells and IgM-expressing cells at day 28.

Claims

1. 1. A method for preparing a population of B cell precursors, comprising: CD34 ＋ contacting a population of hematopoietic stem or progenitor cells (HSPCs) with an induction medium comprising a basal medium and at least one of stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L), and at least one other cytokine; and culturing the population of HSPCs in the induction medium under serum-free conditions to obtain a population of B cell precursors. A method comprising:

2. 2. The method of claim 1, wherein the population of HSPCs is enriched from umbilical cord blood or bone marrow, or is differentiated from pluripotent stem cells (PSCs).

3. The method of claim 1, wherein the population of B cell precursors expresses one or both of CD10 or CD19.

4. The method of claim 1 , wherein the induction medium comprises either SCF or TPO.

5. 5. The method of any one of claims 1 to 4, further comprising contacting the population of B cell precursors with a differentiation medium and culturing the population of B cell precursors in the differentiation medium under serum-free conditions.

6. CD19 ＋ The method of claim 5, further comprising obtaining a population of B lineage cells.

7. After culturing the population of HSPCs in the induction medium, ＋ The method of claim 6, further comprising obtaining B lineage cells.

8. The CD19 ＋ At least a portion of the B lineage cells are IgM ＋ The method of claim 6, which is a cell.

9. The method of claim 5, wherein the differentiation medium comprises a basal medium, at least one of SCF, TPO, and FLT3L, and the at least one other cytokine.

10. The CD19 ＋ contacting a population of B lineage cells with a downstream differentiation medium; and ＋ The method of claim 6, further comprising culturing the population of B lineage cells under serum-free conditions.

11. After culturing the population of B cell precursors in the differentiation medium, ＋ The method of claim 10, further comprising obtaining the cells.

12. The IgM ＋ The method of claim 11 , wherein at least a portion of the cells are antibody-secreting cells.

13. The method of claim 10, wherein the downstream differentiation medium comprises a basal medium, a ligand for human CD40, and the at least one other cytokine.

14. 1. A method for preparing a population of B lineage cells, comprising: contacting the population of B cell precursors with a differentiation medium comprising a basal medium, at least one of SCF, TPO and FLT3L, and at least one other cytokine; and Culturing the population of B cell precursors in the differentiation medium under serum-free conditions to obtain a population of B lineage cells. A method comprising:

15. The method of claim 14, wherein the population of B cell precursors expresses one or both of CD10 or CD19.

16. The population of B cell precursors is either enriched from umbilical cord blood or bone marrow, or differentiated from pluripotent stem cells (PSCs), ＋ The method of claim 14, which is derived from a population of hematopoietic stem or progenitor cells (HSPCs).

17. the population of B lineage cells expressing CD19, and the population of B lineage cells expressing more CD19 than after culturing the population of HSPCs in induction medium to obtain the population of B cell precursors. ＋ The method of claim 16 comprising a cell.

18. CD19 ＋ At least a portion of the B lineage cells are IgM ＋ The method of claim 17, which is a cell.

19. 15. The method of claim 14, further comprising contacting the population of B lineage cells with a downstream differentiation medium and culturing the population of B lineage cells in the downstream differentiation medium under serum-free conditions.

20. After culturing the population of B cell precursors in the differentiation medium, ＋ 20. The method of claim 19, further comprising obtaining the cells.

21. The IgM ＋ 21. The method of claim 20, wherein at least a portion of the cells are antibody-secreting cells.

22. 20. The method of claim 19, wherein the downstream differentiation medium comprises a basal medium, a ligand for human CD40, and the at least one other cytokine.

23. 18. The method of claim 17, wherein the induction medium is serum-free.

24. 24. The method of claim 23, wherein the induction medium comprises a basal medium, at least one cytokine, and one or more of SCF, TPO, and FLT3L.

25. 15. The method of claim 1 or 14, wherein the at least one other cytokine is one or more of IL-3, IL-6, or IL-7.

26. 15. The method of claim 1 or 14, wherein the at least one other cytokine is one or more of IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, or IL-21.

27. The method of claim 1 or 14, wherein the method is carried out under feeder cell-free conditions.

28. The method of claim 27, wherein the feeder cell-free conditions include extracellular matrix proteins or cell adhesion molecules.

29. 29. The method of claim 28, wherein the extracellular matrix protein or the cell adhesion molecule is solubilized or coated onto the surface of a culture vessel.

30. 29. The method of claim 28, wherein the extracellular matrix protein or cell adhesion molecule is fibronectin, vitronectin, laminin, ECM1, SPARC, osteopontin, vascular cell adhesion molecule, immobilized SCF protein, or any combination of the foregoing.

31. 29. The method of claim 28, wherein the feeder cell-free condition is the absence of extracellular matrix proteins or cell adhesion molecules that are solubilized or coated on the surface of the culture vessel.

32. 1. A kit for directed differentiation of B lineage cells, comprising: A basal medium; at least one supplement comprising at least one of stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L), and at least one other cytokine; Including the kit.

33. 33. The kit of claim 32, further comprising a second supplement.

34. 34. The kit of claim 33, wherein the second supplement comprises at least one of stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L), and at least one other cytokine.

35. 35. The kit of claim 33 or 34, wherein the formulation of the at least one supplement is different from the second supplement.

36. 33. The kit of claim 32, further comprising a third supplement.

37. 37. The kit of claim 36, wherein the third supplement comprises a ligand for human CD40 and at least one other cytokine.

38. 33. The kit of claim 32, wherein the at least one cytokine is one or more of IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, or IL-21.

39. A medium for inducing B cell precursors comprising a basal medium, at least one of stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L), and at least one other cytokine.

40. A medium for differentiation of B lineage cells comprising a basal medium, at least one of stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L), and at least one other cytokine.

41. A medium for downstream differentiation of B lineage cells comprising a basal medium, a ligand for human CD40, and at least one other cytokine.

42. 42. The medium of any one of claims 39 to 41, wherein the at least one cytokine is one or more of IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, or IL-21.

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