Large-scale production of iPSC-derived HSCs and progeny

Abstract

The present invention describes a method for the in vitro production of a population of human hemogenic endothelial cells (HECs), which are preferably further differentiated into hematopoietic stem cells (HSCs). In particular, the method relates to differentiating pluripotent stem cell aggregates into hemogenic endothelial cell (HEC) aggregates and further differentiating hemogenic endothelial cell aggregates into hematopoietic stem cell (HSC) aggregates, wherein the step of differentiating PSCs into HECs comprises culturing the cells with BMP4 and culturing the cells without BMP4. The present invention also relates to a population of human HSCs or cryopreserved HSCs, as well as a culture medium comprising the cells or the population of cells, or a bioreactor comprising the culture medium.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention generally relates to methods for the in vitro production of populations of hemogenic endothelial cells (HEC) and / or human hematopoietic stem cells (HSC). In particular, the methods relate to the differentiation of pluripotent stem cell aggregates into hemogenic endothelial cell (HEC) aggregates, and the further differentiation of hemogenic endothelial cell aggregates into hematopoietic stem cell (HSC) aggregates. The present invention further relates to cells comprising a first population of cell aggregates comprising HEC cells and a second population in the form of a single cell suspension comprising HSC cells. The present invention also relates to populations of human HSC or cryopreserved HSC, as well as culture media or bioreactors comprising the cells or populations of cells. [Background technology]

[0002] 2. Background of the Invention The background discussion includes information that may be useful in understanding the present invention. This is not an admission that any of the information provided herein is prior art or important to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Cellular therapy using immune cells expressing chimeric antigen receptors (CARs) has revolutionized the field of cancer. To date, the most effective CAR cell therapy is the treatment of patients with highly relapsed / refractory CD19-positive hematological malignancies using CD19-CAR T cells derived from autologous T cells.

[0004] Despite the medical benefits, several limitations of CAR cell therapy have also become apparent, including difficulties associated with leukapheresis, manufacturing, and efficacy, as well as the high cost and slow turnaround time of adoptive cell therapy (i.e., primary immune cell isolation with subsequent ex vivo manipulation and delivery to patients as a therapeutic).

[0005] To overcome these obstacles, allogeneic CAR strategies have been implemented and have the potential to provide faster, more effective, and more affordable cell therapy.The major obstacle to allogeneic therapy is the tissue compatibility between donor and recipient, which can be mitigated through genome editing approaches to generate a "universal" cell donor and avoid graft-versus-host disease (GVHD), in which transplanted cells recognize host cells as non-self.

[0006] The combination of genome editing and hiPSC technology promises to provide unlimited numbers of allogeneic cells for cell therapy that can be readily available to patients at reduced cost and without the need for long lead times. To meet the clinical demand for allogeneic cell therapy, manufacturing solutions must be developed to incorporate improvements in the use of closed systems, scalability, GMP suitability of starting materials and manufacturing processes, efficacy and reproducibility of differentiation of hiPSCs into specialized lineages, and quality of cells obtained.

[0007] Tursky et al. (Stem Cell Reports, Vol. 15, pp. 735-748, 8 September 2020) describe a head-to-head comparison of different differentiation methods from human induced pluripotent stem cells in 2D or 3D culture systems. Direct hematopoietic derivation of 2D adherent PSCs on special matrices can be successfully achieved, but this would require extremely large surface areas to achieve large-scale commercially useful production. Since 2D culture systems provided the best results in the head-to-head comparison, it is clear that there is an unmet need for alternative efficient suspension cultures, especially those that allow for upscaling to increased yields (e.g., to industrial levels in bioreactors), and methods for providing such.

[0008] WO2020 / 086889A1 discloses a 3D culture method for differentiating pluripotent stem cell aggregates into hemogenic endothelial cell (HEC) aggregates and further differentiating the hemogenic endothelial cell aggregates into hematopoietic stem cell (HSC) aggregates, starting from a plurality of first spheres containing pluripotent stem cells (PSCs), wherein the step of differentiating PSCs into HECs is carried out in the presence of BMP4, inter alia, for the entire duration of the step.

[0009] WO2022019768 relates to infrastructure and associated manufacturing procedures for large-scale production of PSC-derived cells, particularly in closed systems.

[0010] A drawback of the methods described in the art is that they are generally not very efficient, meaning that both the percentage and the absolute number of cells that differentiate into hematopoietic stem cells are relatively low. Another important aspect is that there is a need for improved culture methods that allow the further expansion of hematopoietic stem cells, either immediately after the cells are obtained or after a step of cryopreservation of the obtained cells followed by thawing and culture, so that the cells maintain their characteristics and can further differentiate these cells into specialized lineages, such as T cells and / or NK cells. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO2020 / 086889A1 [Patent Document 2] WO2022019768 [Non-patent literature]

[0012] [Non-Patent Document 1] Tursky et al., Stem Cell Reports, Volume 15, pp. 735-748, September 8, 2020 [Non-Patent Document 2] Chung et al., (2008) Stem Cell Lines, Vol. 2(2): 113 - 117

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Summary of the Invention

Problems to be Solved by the Invention

[0013] In view of this, methods, products and compositions with increased efficiency and yield would be highly desirable, but are not yet readily available. In particular, there is a clear need in the art for reliable, efficient and reproducible methods, products and compositions that allow the differentiation of hematopoietic stem cells to be scaled up for therapeutic purposes. The technical problem underlying the present invention can therefore be seen in the provision of such methods, products and compositions to meet any of the above-mentioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below. [Means for solving the problem]

[0014] SUMMARY OF THE PRESENT APPLICATION As embodied and broadly described herein, the present invention is directed to the surprising finding that when differentiating pluripotent stem cells (PSCs) into HECs, the number of HECs can be dramatically increased when starting with aggregates comprising pluripotent stem cells having an average size of about 20-55 micrometers in diameter, preferably less than about 49 micrometers. It is a further finding that when the differentiation step into HECs starting from aggregated PSCs is performed in a two-step process, in which in a first step BMP4 is added to the medium and in a second step BMP4 is present at a reduced concentration compared to the concentration of BMP4 in the first step, preferably essentially absent from the medium, the formation of definitive hematopoiesis is promoted (as exemplified by reduced CD253a expression on HECs obtained in the methods of the present invention) providing HECs and / or HSCs with high potential for differentiation into both lymphoid and myeloid cell types. Thus, in a first aspect, the present invention provides a method for the in vitro production of a population of hemogenic endothelial cells (HECs), comprising: (a) culturing a suspension of pluripotent stem cells (PSCs), thereby providing a plurality of first cell aggregates comprising said PSCs; (b) culturing the plurality of first cell aggregates in a culture medium for inducing differentiation of PSCs contained in the plurality of first cell aggregates to generate a plurality of second cell aggregates comprising hemogenic endothelial cells (HECs); the plurality of first cell aggregates have an average size of less than or equal to about 49 micrometers in diameter; and / or step (b) comprises a step (b1) of culturing, in which the culture medium comprises a SMAD pathway agonist, preferably BMP4, and a step (b2) of culturing, in which the culture medium is at least partially replaced with fresh medium not comprising a SMAD pathway agonist, and step (b1) is prior to step (b2); Regarding the method: Preferably, the method is carried out in a closed culture system, for example in a bioreactor.

[0015] In another embodiment, the method comprises a step (c) of further differentiating the cells of the second cell aggregate into HSCs using an HSC differentiation medium described herein.

[0016] In a second aspect, the invention relates to a composition comprising a first and a second population of cells, the first population being in the form of cell aggregates comprising HEC cells and the second population being in the form of a suspension of single cells comprising HSC cells, preferably the second population comprising at least 50% HSC cells, or at least 75% HSC cells, or at least 80% or at least 90% HSC, wherein the HSC cells express the markers CD34, CD43 and / or CD45, or the composition being composed of the second population of cells.

[0017] In a third aspect, the present invention relates to a population of human HSCs or cryopreserved HSCs as defined herein.

[0018] In a fourth aspect, the present invention relates to a culture medium or a bioreactor comprising a culture medium comprising a cell or a population of cells as defined herein or comprising cells or aggregates as defined herein.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the present invention are further described herein below with reference to the accompanying drawings. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 shows a schematic representation of the process, biomarkers, and cell culture medium with corresponding growth factors and small molecules. [Diagram 2]Optimization of HEC. Figure 2A: Examples of iPSC aggregates generated using two different iPSC aggregation methodologies. AggreWell plate is a standard academic aggregation technique that works by adding a single cell suspension to a cell culture plate containing microwells, followed by centrifugation to distribute the cells evenly in the microwells. The cells form cell-cell contacts and aggregate together without adhesion to the surface. The bioreactor methodology works by inoculating a single cell suspension into a stirred tank reactor. Small aggregates form spontaneously in the bioreactor due to spontaneous clustering of single cells. The bioreactor methodology is favorable for scalability and does not require manual intervention. Figure 2B: By applying iPSCs to the HEC differentiation process using two different types of aggregate inputs, surprisingly, aggregates generated using the bioreactor methodology lead to approximately 5-fold higher differentiation efficiency of the CD34+ / CD144+ / CD73- target cell population. Figure 2C: Application of T lymphocyte culture conditions to bioreactor-derived HEC aggregates plated on vitronectin-coated tissue culture plastic in XVIVO15 medium + VEGF 50 ng / mL + huSCF 100 ng / mL + bFGF 10 ng / mL + IL7 20 ng / mL) moving forward from day 6 results in a slight upregulation of the lymphoid marker CD1a by day 11. Figure 2D: Subsequent culture in the same culture conditions results in an expansion of this population by 45% after 3 days (day 14), suggesting effective differentiation to the T cell lineage (). Figure 2E: By day 32, more mature T cell markers CD4 and CD3 are robustly expressed in released single cells.FIG. 2F: Application of monocytic cell culture conditions (medium 1 XVIVO15+MCSF 100 ng / mL+IL3 25 ng / mL, or medium 2 XVIVO15+TPO 50 ng / mL+Flt3L 10 ng / mL+huSCF 50 ng / mL+MCSF 80 ng / mL+GMCSF 10 ng / mL) to bioreactor-derived HEC aggregates moving forward from day 6 resulted in pure populations of CD11b, CD14, CD45 in released single cells, exemplary data from day 28 are shown. [Diagram 3] Optimization of aggregate density at the beginning of differentiation to improve process robustness. Fig. 3A,B: To further optimize the process, a shaker deck system based on miniaturized plates in a standard cell culture incubator was used to test how aggregate density affects differentiation of iPSCs to HECs. iPSC aggregates were formed using bioreactor technology and reseeded in plates at the beginning of differentiation. Three densities were tested: 667 aggregates per mL, 6,667 aggregates per mL, and 33,333 aggregates per mL. The relatively low density of 667 aggregates per mL clearly allows the most efficient differentiation, as evidenced by the highest CD34+ / CD144+ / CD73- target cell population. [Figure 4]Figure 4: Optimization of HEC differentiation parameters to enhance HEC to HSC conversion. Hematopoiesis occurs in two waves: primary myeloid-restricted hematopoiesis and secondary lymphomyelopoiesis. CD235a is a key marker that exclusively marks primary hematopoiesis. Figure 4A,B: Further refinement of differentiation conditions using the optimized aggregate method led to the surprising finding that alteration of BMP signaling during the period between iPSC and HEC (days 2-6) was able to significantly reduce CD235a and simultaneously reduce the endothelial marker CD73 without significant effects on the target population CD34+ / CD144+ / CD73-. Figure 4C: Further differentiation of HECs generated by using two different methodologies (with and without BMP4 during days 2-6) resulted in highly pure single cell HSCs when exposed to HSC conditions (IMDM+BSA, ITS-X, β-me, ascorbate-2P, Glutamax, TPO, hSCF, FIT3-L, IL-6 and IL-3) for 4-8 days. Strikingly, cells derived from HECs without BMP4 stimulation on days 2-6 consistently maintain a CD34+ / CD43+ / CD45+ target cell population around 80%, whereas cells derived from days 0-6 continuously exposed to BMP4 conditions rapidly lose target cell fate and upregulate the myeloid marker CD14. Figure 4D: Considering the additional markers CD44 and CD14, it is shown that the improved production method generates HSCs with the expected marker profile. Figure 4E: Repetition of the experiment in the bioreactor and miniaturized shaker plate incubator systems showed similar results with slightly higher cell yields in the bioreactor. Figure 4F: Single cell HSC differentiated from HEC generated using the process without BMP between days 2-6 were harvested, resuspended in CryoStor CS10 and successfully cryopreserved in LN2. Cells were successfully thawed with >90% viability and subsequently exposed to growth conditions (IMDM+BSA, ITS-X, β-me, ascorbate-2P, Glutamax, TPO, hSCF, FIT3-L, IL-6, IL-3 and UM729). Further culture in these conditions led to further expansion proliferation >10-fold.FIG. 4G: Application of NK cell culture conditions to bioreactor-derived HSCs) beginning from day 12 onwards leads to activation of the NK marker CD56 by day 28 of differentiation. [Diagram 5] FIG. 5A: PSC aggregate formation (culture process for 3 days (D-3 to D-O)). FIG. 5B: PSC aggregate formation (culture process for 2 days (D-2 to D-O)). FIG. 5C: Measurement of CD144 / CD73 / CD34 markers in HECs differentiated from PSCs after 2 days of first aggregate formation compared to HECs differentiated from PSCs after 3 days of first aggregate formation. [Figure 6] Figure 1 shows marker expression of primary markers CD235a & KDR (CD309) (%-days of age) per condition tested (2-6, see Table 1). Condition 6 (BMP4 25 ng / mL between D0-D2 and 0 ng / mL between D2-D6) shows reduced CD235 expression compared to the other conditions tested. [Figure 7] Figure 1 shows the fold cell expansion for each of the tested conditions 2-6 (see Table 1) across D0-D6. High BMP4 concentration (100 ng / mL) results in poor yield at day 6, while condition 6 shows good yield at day 6. [Figure 8] FIG. 1 shows the average cell density of suspension cells per mL for each condition 2-6 tested across D3-D14 (see Table 1). [Figure 9] Figure 6 shows marker expression (% age) for each of the conditions tested 2-6 (see Table 1) at days 10, 12 and 14. Conditions (see Figure 6) showing relatively high CD235 expression (primary marker) at day 6, for example, show expression of myeloid marker (CD14) in HSCs at day 14 when BMP4 is added at low concentration (10 ng / mL) but at day 2. Also, high BMP4 concentration (100 ng / mL) results in relatively low yield of HSCs at day 14. [Figure 10]Figure 10A: HSPC purity (% CD45 / CD43 / CD34 expression vs. age), yield (cell concentration vc / mL) and conversion factor (number of CD45+ / CD43+ / CD34+ from one hiPSC). Four different media were tested (see Table 2). Figure 10B: % expression of specific lineage markers CD7 (lymphoid), CD11b (myeloid), CD41 / CD49f vs. age. [Figure 11] Figure 11A: Total cell mass HSPC at day 14 compared to condition 11. Conditions 1, 5, 10 and 11 (see Table 4). Figure 11B: Cell identity (CD34+ or CD34+ / CD7+) at day 14 for conditions 1, 5, 10 and 11 (see Table 4). Figure 11C: CD11b myeloid marker expression at day 14 for conditions 1, 5, 10 and 11 (see Table 4). [Figure 12] Figure 12A: Total cell mass CD34+ / CD7+ cells at day 14 compared to condition 11. Conditions 1, 5, 10 and 11 (see Table 4). Figure 12B: Suspension cell production (vc / mL). Conditions 1, 5, 10 and 11 (see Table 4). [Figure 13] FIG. 1 shows planktonic cell production (vc / mL) for conditions 2, 3, 10, 12, 13 (see Table 5). [Figure 14] FIG. 14A: Cell concentration (vc / mL) CD45+ / CD43+ / CD34+ yield for conditions 2, 3, 10, 12, 13 (see Table 5) at days 10, 12, 14. FIG. 14B: Cell concentration (vc / mL) CD7+ yield for conditions 2, 3, 10, 12, 13 (see Table 5) at days 10, 12, 14. FIG. 14C: Cell concentration (vc / mL) CD11b+ yield for conditions 2, 3, 10, 12, 13 (see Table 5) at days 10, 12, 14. [Figure 15]FIG. 1 shows cumulative HSC expansion under six different post-thaw conditions over time. [Figure 16] FIG. 1 shows CD45+ / CD43+ / CD34+ expression (in %) of cells exposed to six different post-thaw conditions (see Table 6) after 3 and 7 days. [Figure 17] FIG. 1 shows CD7+ lymphoid marker expression (in %) of cells exposed to six different post-thaw conditions (see Table 6) after 3 and 7 days. [Figure 18] FIG. 1 shows CD11b+ myeloid marker expression (in %) of cells exposed to six different post-thaw conditions (see Table 6) after 3 and 7 days. [Figure 19] FIG. 1 shows the amount of CD45+ / CD43+ / CD34+ cells (cell concentration in vc / mL) after 3 and 7 days of exposure to different post-thaw conditions (see Table 6 for details for conditions 2, 5, 6, 7, 8 and 9). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] explanation definition A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspect of this submission for which copyright protection is or may be available in any jurisdiction). The copyright owner has no objection to the copying by anyone of the patent document or the patent disclosure as it appears in the patent file or records at the Patent and Trademark Office, but otherwise reserves all copyrights whatsoever.

[0022] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms should be given their ordinary meaning in the art to which the invention pertains unless otherwise indicated. Other specifically defined terms should be interpreted in a manner consistent with the definitions provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention for testing, the preferred materials and methods are described herein.

[0023] For purposes of the present invention, the following terms are defined below.

[0024] As used herein, the singular terms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0025] As used herein, "about" and "approximately" when referring to measurable values, e.g., amounts, durations in time, etc., are meant to encompass variations of ±20% or ±10%, more preferably ±5%, also more preferably ±1%, and even more preferably ±0.1% from the stated value, where such variations are appropriate to practice the disclosed invention. Unless otherwise clear from the context, all numerical values ​​provided herein include the numerical values ​​modified by the term "about."

[0026] As used herein, "and / or" means that one or more of the specified cases may occur alone or in combination with at least one of the specified cases up to all of the specified cases.

[0027] As used herein, "at least" a particular value means equal to or greater than the particular value. For example, "at least 2" is understood to be the same as "2 or greater", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ... etc. As used herein, the term "up to" a particular value means equal to or less than the particular value. For example, "up to 5" is understood to be the same as "5 or less", e.g., 5, 4, 3, 2, 1, or 0.

[0028] As used herein, "comprising" or "to comprise" is to be interpreted as inclusive and open ended, not exclusive. Specifically, this term and variations thereof mean that the specified features, steps and components are included. These terms should not be understood to exclude the presence of other features, steps or components. This term also encompasses the more restrictive "consisting of."

[0029] As used herein, "conventional techniques" or "methods known to those skilled in the art" means situations in which it would be apparent to a skilled worker how to perform a conventional technique used in the methods of the invention. The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology, and related fields are well known to those of skill in the art and are discussed in various manuals and references.

[0030] As used herein, "exemplary" or "for example" means "serving as an example, instance, or illustration" and should not be construed as excluding other configurations, including those disclosed herein. As used herein, "such as" refers to a specific example of the foregoing.

[0031] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and should not be construed as a limitation on the scope of the invention. The description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range, including both integers and non-integers. For example, the description of a range, e.g., 1-6, should be considered to specifically disclose subranges, e.g., 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, 6, etc. This applies regardless of the breadth of the range.

[0032] As used herein, "aggregates", "aggregation" and "aggregated" in relation to cells refer to one of several major types of cellular structures, namely the connection or clustering of one cell with another cell or cells. Furthermore, it does not include the connection of cells to a substrate, commonly referred to as "adhesion". Cell aggregation is based on cell-cell interactions. Such interactions may form between cells through cell surface proteins and are normally present in many biological systems, e.g., tissues, organs, etc. When compared to single cells, cell aggregation enhances cell survival and function. Cell aggregation can be induced or maintained in vitro by agitating the culture medium containing (pluripotent stem) cells. When agitation of an aqueous suspension of dispersed cells is discontinued, cell aggregates tend to sink (sediment) more quickly to the bottom of the culture vessel than single cells.

[0033] As used herein, "closed culture system" refers to a culture system that includes a culture vessel and added components that are closed / sealed. The closed and / or sealed system is typically sterilized prior to use and after sealing, thus preserving its sterility. During use of the culture vessel, the integrity of the system is not violated, thus maintaining the sterility of the system. Aseptic connections can be made, for example, using single-use sterile connectors or by welding and sealing. The integrity of the system can be violated, for example, by lifting a cap or lid, opening a valve or tube, etc. As used herein, the term "closed system" preferably refers to a closed culture system that includes a culture bioreactor or culture vessel and its components, including a means for mixing the culture medium contained in the culture vessel and a means for withdrawing and exchanging the medium without violating sterility. The bioreactor is used to maintain, cultivate, grow, differentiate, and manipulate cell cultures without violating the sterility integrity of the closed system. An example of a closed culture system suitable for the methods provided herein is described in WO2022019768.

[0034] As used herein, "cryopreservation" refers to a process in which organelles, cells, tissues, extracellular matrices, organs, or any other biological structures susceptible to damage caused by chaotic chemical kinetics are preserved by cooling to very low temperatures. Typically, biological materials are cooled to -80 degrees Celsius or -196 degrees Celsius, thereby stopping any enzymatic or chemical activity that may cause damage to the biological material. Cryopreservation methods aim to reach low temperatures without causing additional damage caused by the formation of ice crystals during freezing. Cryopreservation allows for continued proliferation and / or differentiation of cells after the cryopreserved cells are thawed and provided with a suitable culture medium. Cryopreserved cells are characterized by having a temperature below 0 degrees Celsius, typically below -50, -80, -100, -150, or -180 degrees Celsius.

[0035] As used herein, "culturing", "cultivating", "growing" or variations thereof, when referring to one or more cells, refer to method steps for propagating, expanding or maintaining a population of cells in various types of culture media. Conventional methods and techniques are well known to those skilled in the art of molecular biology, biology, biochemistry, genomics, cell culture, and the like. Although the term "culturing" is generally understood to include cell growth or division, the term also includes methods of differentiating cells in culture media. Growth and differentiation are not mutually exclusive and can occur simultaneously in the same culture medium.

[0036] The term "culture medium" also includes media that are suitable for in vitro cell culture of human or animal cells, preferably over long periods of time. Such culture media contain sufficient ingredients to allow cells to grow, proliferate and / or differentiate over a relatively long period of time, e.g., at least one day. By "defined culture medium" is meant a (growth) medium that is suitable for in vitro cell culture of human or animal cells and in which all of the chemical ingredients are known. Such defined media are free or essentially free of any undefined nutrient sources and / or other undefined factors. Defined culture media may be serum-free.

[0037] As used herein, "differentiating" and "differentiation" refer to the progression of a cell further down a developmental pathway within a lineage. Differentiation of cells, including pluripotent stem cells, can be induced using compounds that direct the differentiation of such stem cells within a lineage. Differentiation is typically controlled by the interaction of cellular genes with the chemical and physical environment of the cell, using signaling pathways that usually involve proteins embedded in the cell surface. Within the context of the present invention, the formation of hemogenic endothelial cells starting from pluripotent stem cells, the formation of hematopoietic stem cells from hemogenic endothelial cells, and the formation of various cells from hematopoietic stem cells, such as erythroid / megakaryocytic progenitor cells, erythrocytes, megakaryocytes, platelets, common lymphoid progenitor cells, lymphoid lineage cells, lymphocytes, natural killer (NK) cells, common myeloid progenitor cells, common granulocyte-monocytic progenitor cells, monocytes, macrophages, dendritic cells, and / or T lymphocytes, including but not limited to regulatory T (Treg) cells (e.g., resting Treg cells, activated Treg cells, or immunosuppressive Treg cells), helper T cells, cytotoxic T cells, memory T cells, natural killer T cells, mucosa-associated invariant T cells, and γδ T cells, are examples of differentiation. The process of differentiation according to the present invention is induced in cells, preferably of human origin, by exposure to a differentiation-inducing culture medium composition and using the methods of the present invention. Differentiation-inducing culture medium compositions typically contain known growth factors that induce differentiation of cells, thereby directing them towards a desired cell type. Differentiation can generally be detected using specific differentiation markers, the presence or absence of which alone or in combination can define the developmental stage of a cell.

[0038] As used herein, "embryonic stem cells", abbreviated as "ES cells" or ESCs (or "hES cells" or "hESCs" if of human origin), refer to stem cells derived from the inner cell mass of a blastocyst. Those skilled in the art will understand methods for obtaining such embryonic stem cells, for example as described by Chung (Chung et al., (2008) Stem Cell Lines, Vol. 2(2):113-117), which utilize techniques that do not cause the destruction of the donor embryo. Various ESC lines are listed in the NIH Human Embryonic Stem Cell Registry.

[0039] As used herein, "hematopoietic stem cells" (HSCs) refer to pluripotent cells that can develop into all types of blood cells, including myeloid and lymphoid lineage cells. HSCs can be found in several organs, e.g., peripheral blood (PB), bone marrow (BM), and umbilical cord blood (UCB), or can be obtained in vitro from pluripotent stem cells. The immediate progeny of hematopoietic stem cells are considered to be "progenitor" cells that can give rise to various cell types within one or more lineages, i.e., erythroid, myeloid, and lymphoid lineages. Hematopoietic stem cells are characterized by the expression of hematopoietic cell surface antigens, such as CD34, CD43, CD45, CD133, or CD117.

[0040] As used herein, "hemogenic endothelial cells" (HECs) refer to specialized vascular endothelial cells that have the potential to give rise to hematopoietic stem cells (HSCs) and / or progenitor cells (HPCs) during vertebrate embryogenesis and / or in vitro upon differentiation. During this differentiation process, sometimes referred to as endothelial hematopoietic transformation (EHT), HECs gradually round up, separate and bud off from their neighbors, thereby releasing HSCs as single cells into the environment. Human markers for HECs include CD31, CD144, CD34, and CD184.

[0041] As used herein, "in vivo" refers to events that occur within a subject's body; "in vitro" refers to events that occur outside of a subject's body. For example, an in vitro assay or method includes any assay or method that is performed outside of a subject. An in vitro assay or method includes cell-based assays, in which living or dead cells are utilized. An in vitro assay also includes cell-free assays, in which intact cells are not utilized.

[0042] As used herein, "induced pluripotent stem cells" or "iPSCs" refer to pluripotent stem cells derived from cells that are not pluripotent stem cells (i.e., cells that are differentiated relative to pluripotent stem cells). Induced pluripotent stem cells can be derived from a number of different cell types, including terminally differentiated cells. Induced pluripotent stem cells generally have an embryonic stem cell-like morphology, growing as flat colonies with a large nuclear-cytoplasmic ratio, clear borders, and prominent nuclei. In addition, induced pluripotent stem cells may express one or more key pluripotency markers known by those skilled in the art. To generate induced pluripotent stem cells, somatic cells can be provided with reprogramming factors (e.g., OCT3 / 4, SOX2, KLF4, MYC, NANOG, LIN28, etc.), which are known in the art to reprogram somatic cells to become pluripotent stem cells.

[0043] As used herein, the term "marker" is used to describe a characteristic and / or phenotype of a cell. Markers can be used for the selection of cells containing a characteristic of interest. Markers will vary with the particular cell. A marker is a characteristic, whether a morphological, functional or biochemical characteristic of a cell of a particular cell type, or a molecule expressed by a cell type. Preferably, such a marker is a protein, more preferably carrying an epitope for an antibody or other binding molecule available in the art. However, markers can consist of any molecule found in cells, including but not limited to proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids. Examples of morphological markers include shape, size, and nucleocytoplasmic ratio. Examples of functional markers include the ability to migrate under certain conditions and the ability to differentiate along a particular lineage. Markers can be detected by any method available to the skilled artisan. A marker can also be the absence of a morphological characteristic or the absence of a protein, lipid, etc. A marker can be a combination of the intrinsic characteristics of the presence and absence of polypeptides as well as a panel of other morphological characteristics.

[0044] As used herein, "pluripotent stem cells" or "PSCs" refer to stem cells capable of generating a wide range or variety of cell types of an organism, capable of generating cells of mammalian germ layers, e.g., endoderm, mesoderm, and ectoderm, and encompass at least pluripotent embryonic stem cells and induced pluripotent stem cells. Pluripotent stem cells can be obtained in various ways. Induced pluripotent stem cells (iPSCs) can be derived, for example, from somatic cells. Pluripotent stem cells may also be in the form of established cell lines. According to an embodiment, PSCs for use in the methods according to the invention are derived from human cells or tissues, e.g., iPSCs for use in the methods described herein can be derived from human cells, e.g., human somatic cells. Such human PSCs or human iPSCs may also be referred to as hPSCs or hiPSCs, respectively. The progeny of PSCs or iPSCs (e.g., HECs or HSCs), when derived from cells of human origin, may equally be given the prefix "human" or "h" and are equally considered for the methods described herein.

[0045] PSCs, e.g. iPSCs, for use according to the present invention may be further modified, e.g. by transfection with an expression vector encoding a functional protein, such as but not limited to a chimeric antigen receptor, a signaling peptide, a peptide required by a subject in need of protein replacement therapy to replace a (genetic) defective, abnormal or absent functional protein in the subject.

[0046] As used herein, "proliferating" and "proliferation" refer to an increase in the number of cells in a population (growth) by cells undergoing cell division, i.e., mitosis. Cell proliferation is generally understood to result from the coordinated activation of multiple signaling pathways in response to the environment, including growth factors and other mitogens. Cell proliferation may also be promoted by the action of intracellular or extracellular signals and release from mechanisms that inhibit or negatively affect cell proliferation.

[0047] As used herein, "stem cells" refers to a population of undifferentiated cells defined by their ability at the single-cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitor cells, non-replicating progenitor cells, and terminally differentiated cells (Morrison et al., (1997) Cell 88:287-298).

[0048] Detailed Description The embodiments of the present invention are defined herein. It is considered that any method, use or composition described herein can be carried out with respect to any other method, use or composition described herein. The embodiments discussed in the context of the method, use and / or composition of the present invention can be utilized with respect to any other method, use or composition described herein. Thus, the embodiments pertaining to one method, use or composition can be applied to other methods, uses and compositions of the present invention as well.

[0049] Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions and medicaments of the invention for use in a method for treatment of the human (or animal) body by therapy.

[0050] As embodied and broadly described herein, the present invention is directed to an improved method for differentiating pluripotent stem cells into hemogenic endothelial cells (HECs) (steps (a) and (b)) and / or HECs (further) differentiated into hematopoietic stem cells (step (c)). The method is for inducing differentiation of pluripotent stem cells into HECs, which according to further embodiments are cultured into hematopoietic stem cells (HSCs), and / or for the production of HECs derived from such differentiated pluripotent stem cells and / or HSCs derived from such differentiated pluripotent stem cells. The method can be performed, for example, in a closed culture system, in particular without an intermediate cell selection step. The method allows for the production of huge amounts of such differentiated cells (i.e. HECs and / or HSCs). The method allows for a high output of cells over the input of the cell ratio (e.g. expressed by the number of cells). The methods also allow for the generation of HSCs that can be further expanded several fold (e.g., 2, 3, 5, 10, 20 or more), either directly or after cryopreservation and thawing, and subsequently differentiated (e.g., using differentiation protocols as described in the art) into specialized lineages as described herein.

[0051] The present invention takes advantage of existing 3D cell culture protocols for differentiation of pluripotent stem cells. The present invention provides improved methods that allow to increase the efficiency and yield of the method for generating HECs and / or HSCs. The efficiency of differentiation of pluripotent stem cells into HECs and / or differentiation of HECs into HSCs relates, for example, to the percentage of cells in the aggregates that eventually become HSCs and also to the percentage of cells that do not become HSCs as a result, for example, by differentiating into a different (undesirable or less desirable) cell type. The yield, as measured in absolute cell numbers (HECs and / or HSCs) generated by differentiation of pluripotent stem cell aggregates as defined for the methods herein, depends on the efficiency of the differentiation process and also on the proliferation of cells during the expansion protocol. In particular, it has been found that the current methods for in vitro production of HECs and / or HSCs can be suitably used in high-volume closed culture systems, for example using culture vessels, such as bioreactors, tanks, or any other device suitable for the culture of cells. For example, the volume of the culture vessel may be any volume, but is preferably 2-150 liters, or 2-100 liters, or 2-50 liters, and / or allows for culture in such volumes of culture medium. For example, the method of the invention allows for culture in at least 2, 3, 5, 8, 10, 20, 50 liters of culture medium. In a further embodiment, the efficiency relates to the marker profile of the HECs and / or HSCs resulting from the method. The marker profile of the HECs and / or HSCs will then determine the potential of the cells for further differentiation in specialized hematopoietic cell types. The differentiation protocol of the aggregated PSCs into HECs as described herein with respect to step (b) promotes secondary lympho-myeloid hematopoiesis. The formation of (high purity) secondary hematopoietic HECs and / or HSCs as described herein allows for efficient differentiation of the HECs and / or HSCs into downstream lymphoid or myeloid cell types, thereby reducing the need for intermediate cell type-based isolation and / or selection steps.

[0052] Thus, in a first aspect, the present invention provides a method for the in vitro production of a population of hemogenic endothelial cells (HECs), comprising: (a) culturing a suspension of pluripotent stem cells (PSCs), thereby providing a plurality of first cell aggregates comprising said PSCs; (b) culturing the plurality of first cell aggregates in a culture medium for inducing differentiation of PSCs contained in the plurality of first cell aggregates to generate a plurality of second cell aggregates comprising hemogenic endothelial cells (HECs); - the plurality of first cell aggregates have an average size of less than or equal to about 49 micrometers in diameter; and / or - step (b) comprises a step (b1) of culturing, in which the culture medium comprises a SMAD pathway agonist, preferably BMP4, and a step (b2) of culturing, in which the culture medium is at least partially replaced by fresh medium not comprising a SMAD pathway agonist, and step (b1) is prior to step (b2); It concerns the method.

[0053] In some embodiments, the fresh medium in step (b2) is not supplemented with a SMAD pathway agonist.

[0054] In one embodiment, the present invention provides a method for the in vitro production of a population of hemogenic endothelial cells (HECs), comprising: (a) culturing a suspension of pluripotent stem cells (PSCs), thereby providing a plurality of first cell aggregates comprising said PSCs; (b) culturing the plurality of first cell aggregates in a culture medium for inducing differentiation of PSCs contained in the plurality of first cell aggregates to generate a plurality of second cell aggregates comprising hemogenic endothelial cells (HECs); - the plurality of first cell aggregates have an average size of about 49 micrometers or less in diameter; It concerns the method.

[0055] In another embodiment, the present invention provides a method for the in vitro production of a population of hemogenic endothelial cells (HECs), comprising: (a) culturing a suspension of pluripotent stem cells (PSCs), thereby providing a plurality of first cell aggregates comprising said PSCs; (b) culturing the plurality of first cell aggregates in a culture medium for inducing differentiation of PSCs contained in the plurality of first cell aggregates to generate a plurality of second cell aggregates comprising hemogenic endothelial cells (HECs); step (b) comprises a step (b1) of culturing, in which the culture medium comprises a SMAD pathway agonist, preferably BMP4, and a step (b2) of culturing, in which the culture medium is at least partially replaced with fresh medium not comprising a SMAD pathway agonist, and step (b1) is prior to step (b2); It concerns the method.

[0056] In another embodiment, the present invention provides a method for the in vitro production of a population of hemogenic endothelial cells (HECs), comprising: (a) culturing a suspension of pluripotent stem cells (PSCs), thereby providing a plurality of first cell aggregates comprising said PSCs; (b) culturing the plurality of first cell aggregates in a culture medium for inducing differentiation of PSCs contained in the plurality of first cell aggregates to generate a plurality of second cell aggregates comprising hemogenic endothelial cells (HECs); - the plurality of first cell aggregates have an average size of less than or equal to about 49 micrometers in diameter; and - step (b) comprises a step (b1) of culturing, in which the culture medium comprises a SMAD pathway agonist, preferably BMP4, and a step (b2) of culturing, in which the culture medium is at least partially replaced by fresh medium not comprising a SMAD pathway agonist, and step (b1) is prior to step (b2); It concerns the method.

[0057] It is a common understanding in the field of stem cell differentiation that the presence of a SMAD pathway agonist, preferably BMP4, in the culture medium is required during the differentiation step from PSC to HEC in order to differentiate pluripotent stem cells into HEC (and optionally further into HSC). It has now been surprisingly found by the inventors that unexpectedly, the yield and efficiency of hematopoietic stem cell generation can be increased only if a SMAD pathway agonist, preferably BMP4, is provided in the culture medium during a portion of the differentiation step from PSC to HEC. In particular, it has been found that a first step (b1) of culture in which a SMAD pathway agonist, preferably BMP4, is added to the culture medium and a subsequent step (b2) of culture in which no SMAD pathway agonist is added to the culture medium increases the differentiation efficiency and yield. For example, step (b1) can be performed for 1, 2, 3, or 4 days, preferably about 2 days, followed by step (b2) for 1, 2, 3, 4, 5, or 6 days, preferably about 4 days. In a further embodiment, the entirety of step (b) is carried out over a period of about 6 days.

[0058] Moreover, it has surprisingly been discovered that when the size of the first cell aggregates is carefully controlled, particularly when starting with aggregates containing pluripotent stem cells having an average size of about 49 micrometers or less in diameter, the yield and efficiency of HEC and / or subsequent HSC generation can be unexpectedly increased.

[0059] In particular, differentiation into HECs and / or HSCs consists of a delicate and balanced refinement of different (signaling) pathways that are turned on or off at different stages of differentiation, and without wishing to be bound by theory, the inventors believe that by initiating differentiation of PSCs upon formation of aggregates having an average size of about 49 micrometers in diameter or less, the cells within the aggregates can more efficiently proliferate and differentiate into HECs and thereafter into HSCs, as defined and described herein, thereby providing a novel and robust method of producing a population of cells comprising HECs and / or HSCs in high absolute and / or relative amounts (purity), where the HSCs can be efficiently further propagated (or expanded), either directly or after cryopreservation and thawing. Surprisingly, it has been found that HSCs obtained by the methods described herein can be expanded several fold as described above while substantially maintaining their HSC phenotype, thus enabling subsequent differentiation of HSCs into more specialized cell lines in large numbers, high purity and / or high yield.

[0060] In a further embodiment, the present invention provides a method for producing a method for producing a cell aggregate comprising the steps of: (a) culturing a suspension of pluripotent stem cells (PSCs), thereby providing a plurality of first cell aggregates comprising said PSCs; (b) culturing the plurality of first cell aggregates in a culture medium for inducing differentiation of PSCs contained in the plurality of first cell aggregates to generate a plurality of second cell aggregates comprising hemogenic endothelial cells (HECs), wherein step (b) comprises a step (b1) of culturing, wherein the culture medium comprises a SMAD pathway agonist, preferably BMP4, and at least a partial step of culturing, wherein the culture medium comprises a fresh medium not comprising a SMAD pathway agonist. and a step (b2) of culturing the plurality of second cell aggregates in a culture medium for inducing differentiation of HECs contained in the plurality of second cell aggregates to generate a plurality of third cell aggregates that generate hematopoietic stem cells (HSCs), and allowing HSCs to be released (via budding) from the plurality of third cell aggregates in a culture medium for obtaining a population of HSCs, thereby forming a single cell population comprising HSCs in suspension in the culture medium.

[0061] Further embodiments for carrying out steps (a), (b) and (c) according to the present invention are provided herein below and can be combined independently to provide specific embodiments of the method of the present invention.

[0062] A non-limiting exemplary scheme for differentiation of pluripotent cells into HECs and / or HSCs can be found in FIG. 1. For example, pluripotent stem cells, e.g., induced pluripotent stem cells, can be cultured under appropriate conditions. Suitable culture conditions for expanding pluripotent stem cells are known to those skilled in the art. For example, mTeSR1 medium can be used with vitronectin culture plate coating and passaging with Accutase, but those skilled in the art will be aware of other suitable media and / or additives. Cells obtained from undifferentiated pluripotent stem cell culture can be used to inoculate 3D culture, e.g., in a bioreactor. After inoculation, cell aggregates of pluripotent stem cells will start to form. Aggregate formation can be induced, for example, by selecting appropriate culture conditions, and in a preferred embodiment, StemMACS™ iPS-Brew XF medium can be used, for example, preferably in the presence of a ROCK inhibitor, e.g., preferably Y-27632. Those skilled in the art will know and understand that in addition to those described herein, other suitable culture media, such as mTeSR1, Essential 8, TeSR E8, and / or Nutristem media, can be used as well. Those skilled in the art will also know and understand that alternative additives, such as other ROCK inhibitors, such as blebbistatin, fasudil, pinacidil, thiazovivin, chroman-1, and / or molecules with similar survival-promoting potency, such as emricasan, fasudil, thiazovivin, Q-VD-Oph, polyamines, and / or trans-ISRIB, can be used. After aggregate formation, the aggregates containing pluripotent stem cells are differentiated into HEC-containing aggregates, followed in further embodiments by further differentiation into aggregates that generate HSCs. Individual HSCs can be released from the aggregates and isolated, for example, for cryopreservation and subsequent use, such as further expansion and subsequent differentiation.

[0063] In step (a), a plurality of first cell aggregates comprising pluripotent stem cells (PSCs) are provided. Provided herein are embodiments further defining the first cell aggregates for differentiation into HECs according to the method of the present disclosure. In some embodiments, the plurality of first cell aggregates have an average size of 49 micrometers or less in diameter.

[0064] Depending on the number of pluripotent stem cells to be introduced, it may be decided to initially grow the pluripotent stem cells for a certain period of time to obtain a desired number of pluripotent stem cells (aggregates), for example until the majority of the first cell aggregates have a desired average size in diameter. As mentioned above, the culture medium can be any suitable culture medium for the growth of pluripotent stem cells, for example the commercially available mTeSR1, StemMACS™ iPS-Brew XF, Essential 8, TeSR E8, mTeSR Plus and / or Nutristem medium. In a (preferred) embodiment in which the pluripotent stem cells are initially introduced as a single cell suspension, the single cells will form aggregates during the culture in the culture medium for the growth of the stem cells (aggregation typically begins within a few hours, for example after 2-3 hours).

[0065] In an embodiment, the PSCs in the plurality of first cell aggregates express the markers OCT3 / 4, SOX2, and NANOG in at least 80% of the cells. In an embodiment, the PSCs are induced pluripotent stem cells.

[0066] In some embodiments, the plurality of first cell aggregates have an average size of about 20-250 micrometers in diameter, or an average size of about 20-150 micrometers, preferably an average size of about 20-55 micrometers, or an average size of about 25-50 micrometers, or an average size of about 30-40 micrometers.

[0067] In some embodiments, the first cell aggregates have an average size of about 49 micrometers or less in diameter. In one non-limiting example, an average size of about 20-55 micrometers in diameter, or an average size of about 49 micrometers or less, can be achieved about 2 days after inoculation, e.g., 12-96 hours after inoculation, preferably 24-72 hours after inoculation, typically resulting in a density of about 110,000 aggregates per mL. Those skilled in the art will know and understand how to determine the diameter of cell aggregates, e.g., using methods as described herein.

[0068] Additionally, in embodiments, the plurality of first cell aggregates have an average size of about 25-50 micrometers, or have an average size of about 30-45 micrometers.

[0069] It has surprisingly been found that in the process of the present invention, when starting with aggregates comprising pluripotent stem cells having an average size of about 20-55 micrometers in diameter, preferably having an average size of about 49 micrometers or less, or about 45 micrometers or less, the yield and efficiency of hematopoietic stem cell generation can be unexpectedly increased.

[0070] In particular, differentiation into HECs and further into HSCs consists of a delicate and balanced refinement of different (signaling) pathways that are turned on or off at different stages of differentiation, and without wishing to be bound by theory, the inventors believe that by selecting aggregates comprising pluripotent stem cells having an average size of about 20-55 micrometers in diameter, preferably having an average size of about 49 micrometers or less, or about 45 micrometers or less in diameter, the cells within the aggregates can be efficiently expanded and differentiated into HECs and thereafter into HSCs, as defined and described herein, thereby providing a novel and robust method of producing a population of cells comprising HSCs in high absolute and relative quantities, where the HSCs can be efficiently further expanded (or expanded), either directly or after cryopreservation and thawing. Surprisingly, it has been found that HSCs can be expanded several fold as described above while substantially maintaining the HSC phenotype, thus allowing the subsequent differentiation of HSCs into more specialized cell lines in large numbers and high yields.

[0071] For example, the aggregate size of the plurality of first cell aggregates comprising PSCs may be 20, 21, 22, 23, 23, 24, 25, 27, 28, 29, or 30 micrometers or more, or the aggregate size of the plurality of first cell aggregates comprising PSCs may be 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, or 45 micrometers or less, preferably about 49, 48, 47, 46, or 45 micrometers or less in diameter. Since the aggregates are substantially spherical, size as used herein refers to diameter, and the diameter is defined as the longest straight line between two points on the surface of the aggregate. Furthermore, since the methods and compositions refer to a plurality of aggregates, it is understood that aggregate size refers to the average size of the aggregates in the culture medium. It is therefore understood that a certain percentage of the aggregates, for example 25%, 20%, 15%, 10%, preferably 5% or less, may be either larger or smaller than the indicated size ranges. Thus, the average aggregate size of the plurality of first cell aggregates comprising PSCs may be 20-55 micrometers, preferably 22-52, 25-50, 25-49, 28-48, or 30-45 micrometers.

[0072] The inventors have further surprisingly found that aggregate density, as measured as aggregates per mL, can further affect the efficiency of differentiation, as described herein. Representative data is shown in Figure 3, comparing different experiments using three different aggregate densities: 667, 6667 and 33333 aggregates per mL. An otherwise identical protocol was used to differentiate the aggregates, which were tested for the markers CD73, CD144 and CD34 on day 7, and CD45 on day 21. As can be observed from Figure 3B, where differentiation under conditions of 6667 or 667 aggregates per mL resulted in fewer CD73 positive cells and more CD144 and CD34 positive cells on day 7, and more CD45 positive cells on day 21, when compared to differentiation with 33333 aggregates per mL, a substantial increase in differentiation efficiency can be observed when decreasing the aggregate concentration from 33333 to 6667 aggregates.

[0073] Thus, in an embodiment, the plurality of first cell aggregates are provided in the first culture medium at a density of at least 100 aggregates / mL, preferably 100 to 100000 aggregates / mL. For example, the plurality of first cell aggregates are provided in the first culture medium at a density of at least 100, 150, 200, 250, 300, 350, 400, 450, or 500 aggregates / mL, and / or at a density of no more than 100000, 90000, 80000, 70000, 60000, 50000, 40000, 30000, 25000, 20000, 18000, 16000, 15000, 14000, 13000, 12000, 11000, 10000, 9000, or 8000 aggregates / mL. Thus, a plurality of first cell aggregates are provided in a first culture medium at a density of 100-100,000, 150-60,000, 200-20,000, 250-10,000, 250-8,000, 250-6,000, 250-4,000, or 250-2,000 aggregates / mL.

[0074] In step (b), the plurality of first cell aggregates are cultured in a culture medium for inducing differentiation of PSCs contained in the plurality of first cell aggregates to generate a plurality of second cell aggregates containing hemogenic endothelial cells (HECs). In some embodiments, step (b) comprises a step (b2) of culturing in which the culture medium does not contain a SMAD pathway agonist, such as BMP4, and preferably, step (b) comprises a step (b1) of culturing in which the culture medium contains a SMAD pathway agonist, preferably BMP4, and a step (b2) of culturing in which the culture medium is at least partially replaced with a fresh medium that does not contain a SMAD pathway agonist, preferably BMP4, and step (b1) is before step (b2).

[0075] During step (b), the pluripotent stem cells in the first cell aggregates differentiate at least in part into HECs. Suitable culture conditions for differentiating pluripotent stem cells into HECs are known to those skilled in the art. For example, IF9S medium can be used, which is an IMDM / F12-based medium containing nine additives as described in Uenishi et al., Stem Cell Reports. 2014 Dec. 9;3(6):1073-1084. Typically, the culture medium is further supplemented with suitable growth factors, such as, but not limited to, VEGF (e.g., 5-125 ng / mL, preferably about 25 ng / mL), bFGF (e.g., 5-125 ng / mL, preferably about 25 ng / mL), BMP4 (e.g., 5-125 ng / mL, 5-50 ng / mL, 10-40 ng / mL, 10-30 ng / mL, or preferably about 25 ng / mL), CHIR99021 (e.g., 0.6-15 μM, preferably about 3 μM), and SB431542 (e.g., 0.6-15 μM, preferably about 3 μM). Non-limiting examples of other suitable growth media for step (b) are StemPro, Hams-F12, APEL, and RPMI. Non-limiting examples of suitable additives are KO-SR, N-2 supplement, and B-27 supplement.

[0076] In some embodiments, the culture medium comprises VEGF and / or bFGF throughout step (b), including, for example, steps (b1) and (b2).

[0077] Other suitable growth factors for step (b) include, but are not limited to, Wnt3a, SCF, Flt3-L, TPO.

[0078] In an embodiment, step (b) is carried out for about 6 days, such as, for example, 3, 4, 5, 6, 7, 8, 9, or 10 days or more. Preferably, step (b) is carried out for 2 to 10 days, preferably 3 to 8 days, or preferably 5 to 6 days.

[0079] In an embodiment of the method of the present invention, a plurality of first cell aggregates are obtained by suspension culture in a stirred tank bioreactor. In an embodiment, a plurality of second cell aggregates are present in a second culture medium at a density of 200,000-800,000 cells / mL. In an embodiment, at least 10-50%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the cells in the plurality of second cell aggregates are HECs.

[0080] In embodiments, the culture medium during step (b) is replaced every day, every other day, every 3 days, every 4 days, or a combination thereof (e.g., the first replacement after 1 day and the subsequent replacement after 2 days). In certain preferred embodiments, the culture medium during step (b) is replaced or refreshed every day.

[0081] In some embodiments, during step (b), including steps (b1) and (b2) as described below, at least a portion of the medium, preferably the entire medium, is replaced or renewed. However, it is noted that it has surprisingly been found that it is possible to design a robust and reproducible manufacturing process, preferably in and with a closed culture system, that results in a large amount of well-differentiated cells, not by withdrawing the entire culture medium from the culture vessel and replacing it with fresh culture medium, but by withdrawing only a portion of the culture medium from the culture vessel before the subsequent fresh culture medium (and, if necessary, growth factors, such as those disclosed herein) are added to the remaining culture medium in the culture vessel (and thus the portion of the previous medium is mixed with the subsequent medium). For example, in one embodiment, up to 95 vol.%, 90 vol.%, 85 vol.%, or 80 vol.% of the culture medium in the culture vessel is withdrawn, thereby allowing an efficient switch of medium composition and / or the recovery of (single) cells or substances secreted by the cells into the culture medium. For example, in this embodiment, if the culture vessel contains 10 liters of culture medium, up to 9500, 9000, 8500 or 8000 milliliters of culture medium are withdrawn from the culture vessel.

[0082] At the same time, it has been found that preferably at least 30 vol.%, 40 vol.%, 50 vol.%, 60 vol.% or 70 vol.% of the culture medium is recovered from the culture vessel. Thus, in some embodiments, for example, 30 vol.% to 95 vol.%, or 40 vol.% to 95 vol.%, or 50 to 95 vol.%, or 60 to 90 vol.%, or 60 to 80 vol.%, or 70 to 90 vol.%, or 70 to 80 vol.% of the culture medium is recovered from the culture vessel and preferably replaced or renewed with new culture medium. As will be understood by the skilled artisan, the amount or percentage of medium recovered may vary between different moments of medium recovery according to the method of the present invention. For example, during a first medium withdrawal in step (b), 70 vol.% of the culture medium in the culture vessel can be withdrawn and replaced, and during a subsequent medium withdrawal in step (b), the same, more, or less medium (e.g., 50 vol.% or 75 vol.%) can be withdrawn and replaced. In some embodiments, growth factors are added and provided directly to the culture medium already present in the culture vessel or closed culture system, without substantial replacement of old culture medium or substantial addition of new culture medium (e.g., fresh medium).

[0083] In some embodiments, between steps (b1) and (b2), at least 70 vol.%, 80 vol.%, 90 vol.% or 95 vol.% of the medium is replaced with fresh medium. In some embodiments, partially replacing the culture medium comprises replacing at least 30 vol.%, at least 40 vol.%, at least 50 vol.%, at least 60 vol.%, 70 vol.%, 80 vol.%, 90 vol.% or 95 vol.% of the medium with fresh medium.

[0084] In embodiments, the duration of step (b2) is more than 50% of step (b) and / or the duration of step (b1) is less than 50% of step (b), preferably step (b2) lasts for at least 2, 3 or 4 days and / or step (b1) lasts for at most 1, 2, 3 or 4 days. For example, step (b1) is 1 day and step (b2) is 2 days, step (b1) is 1 day and step (b2) is 3 days, step (b1) is 1 day and step (b2) is 4 days, step (b1) is 1 day and step (b2) is 5 days, step (b1) is 1 day and step (b2) is 6 days, step (b1) is 2 days and step (b2) is 3 days, step (b1) is 2 days and step (b2) is 4 days, step (b1) is 2 days and step (b2) is 5 days. or step (b1) is 2 days and step (b2) is 6 days, or step (b1) is 3 days and step (b2) is 4 days, or step (b1) is 3 days and step (b2) is 5 days, or step (b1) is 3 days and step (b2) is 6 days, or step (b1) is 3 days and step (b2) is 7 days, or step (b1) is 4 days and step (b2) is 5 days, or step (b1) is 4 days and step (b2) is 6 days, or step (b1) is 4 days and step (b2) is 7 days.

[0085] In some embodiments, the duration of step (b2) is more than 50% of step (b) and / or the duration of step (b1) is less than 50% of step (b), preferably, step (b2) lasts for at least 2, 3 or 4 days (e.g., 4 days) and / or step (b1) lasts for at most 3, 2 or 1 days (e.g., 2 days).

[0086] It is understood that BMP4 is a TGF-β family growth factor, and therefore it is envisaged that in step (b) an alternative TGF-β family growth factor or SMAD pathway agonist may be added to the culture medium. In such a case, in step (b1), preferably the cells are cultured in a medium containing a TGF-β family growth factor or SMAD pathway agonist. Thus, in an embodiment, the step of culturing a plurality of first cell aggregates in step (b) includes a step (b2) of culturing in which a TGF-β family growth factor or a SMAD pathway agonist is not added to the culture medium, and preferably, the step of culturing a plurality of first cell aggregates in step (b) includes a step (b1) of culturing in which the culture medium contains a TGF-β family growth factor, or a SMAD pathway agonist, and / or a GSK3 inhibitor, and a step (b2) of culturing in which a TGF-β family growth factor, or a SMAD pathway agonist, and / or a GSK3 inhibitor is not added to the culture medium, and step (b1) is before step (b2).

[0087] The TGF-β family growth factor can be selected from TGFB1, TGFB2, TGFB3, BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11, BMP15, GDF1, GDF2, GDF3, GDF5, GDF6, GDF8, GDF9, GDF10, 11, GDF15, INHA, INHBA, INHBB, INHBC, INHBE, activin A, activin AB, activin B, LEFTY1, LEFTY2, MSTN, nodal, NRTN, PSPN, AMH, ARTN, or combinations thereof. Alternatively, the TGF-β pathway can also be activated by small molecule agonists, such as, but not limited to, SRI-011381 hydrochloride. The GSK3 inhibitor can be selected from beryllium cation, copper cation, lithium cation, mercury cation, tungsten cation, 6-BIO, dibromocantalerin, hymenialdesine, indirubin, meridianin, CHIR99021, CT98014, CT98023, CT99021, TWS119, SB-216763, SB-41528, AR-A014418, AZD-1080, alsterpaullone, cazpaullone, kenpaullone, manzamine A, palinurine, tricanthin, TDZD-8, NP00111, NP031115, tideglusib, HMK-32, L803-mts, L807-mts, COB-187, COB-152, or a combination thereof.

[0088] In further embodiments, during at least a portion of step (b2), the cells are cultured in the presence of a TGF-β pathway inhibitor, such as SB431542 (e.g., 0.6-15 μM, preferably about 3 μM). Alternatives to SB431542 contemplated for use in step (b2) of the methods herein include other Activin / Nodal / TGF-β pathway inhibitors known to those of skill in the art, such as A83-01.

[0089] In a further embodiment, during at least a portion of step (b2), the cells are also cultured in the presence of a GSK3 inhibitor, preferably a GSK3-β inhibitor, such as CHIR99021 (e.g., 0.6-15 μM, preferably about 3 μM), or a combination thereof. Alternatives to CHIR99021 contemplated for use in step (b2) of the methods herein include other GSK3-β inhibitors or other more general compounds known to those skilled in the art to activate the Wnt pathway, such as, but not limited to, the list of compounds in Table 1 as published by Bonnet C. et al., 2021 ("Wnt signaling activation: targets and therapeutic opportunities for stem cell therapy and regenerative medicine" RSC Chem Biol. 2021 Aug. 5; 2(4): 1144-1157).

[0090] As explained above with respect to step (b), as part of steps (b1) and (b2), all the medium can be replaced or renewed. However, it has surprisingly been found that it is possible to design a robust and reproducible manufacturing process that produces a large amount of well-differentiated cells in a closed culture system, not by withdrawing the entire culture medium from the culture vessel and replacing it with fresh culture medium, but by withdrawing only a portion of the culture medium from the culture vessel before the subsequent fresh culture medium (and, if necessary, growth factors, such as those disclosed herein) are added to the remaining culture medium in the culture vessel (and thus the portion of the previous medium is mixed with the subsequent medium). In these embodiments, it is understood that residual medium is still present, for example, in some culture conditions, up to 20% or more of the medium can remain at the time when the fresh medium is added. The remaining medium (e.g., in step (b1)) may still contain some BMP4 (or TGF-β family growth factor, or SMAD pathway agonist), and therefore, when "cultured in a medium without BMP4" or "a culture step in which BMP4 is not added to the culture medium" (or TGF-β family growth factor, or SMAD pathway agonist) is referred to herein, it is intended that in this step, the new medium added does not contain BMP4 (or TGF-β family growth factor, or SMAD pathway agonist). In other words, step (b2) involves the replacement of all or part of the medium contained in the culture system with fresh medium without BMP4. When only a part of the medium in the culture system is replaced or renewed, residual old culture medium is still present. Unexpectedly, it has been found that the inclusion of step (b2) increases differentiation efficiency and yield. In particular, as shown in the Examples, it has been found that HSCs so obtained can be efficiently expanded, e.g., after cryopreservation and thawing, without substantial loss of their phenotypic properties (see Examples).

[0091] In some embodiments, the culture medium used for culturing according to step (b2) comprises (residual) less than 10 ng / mL, preferably less than 5, 4, 3, 2, 1 ng / mL or 0 ng / mL BMP4.

[0092] In some embodiments, the SMAD pathway agonist is BMP4, and the concentration of BMP4 in the culture medium in step (b1) is 10 to 40 ng / mL, for example 10 to 30 ng / mL or about 25 ng / mL, and / or the (residual) concentration of BMP4 in the culture medium in step (b2) is less than 5 ng / mL.

[0093] In certain embodiments, a method is provided for the production of HECs, comprising step (a) culturing a suspension of PSCs, thereby providing a plurality of first cell aggregates comprising said PSCs; step (b1) culturing the first cell aggregates obtained in step (a) in the presence of a SMAD agonist, in particular BMP4, VEGF and bFGF, and step (b2) comprising a subsequent step of culturing the cells obtained in step (b1) in the presence of each of VEGF, bFGF, CHIR99021 and SB431542 during at least a portion of step (b2), wherein the culture medium during step (b2) does not comprise a SMAD pathway agonist (i.e., no SMAD pathway agonist is added to the culture medium for use in said step (b2)), and wherein between steps (b1) and (b2) the culture medium is replaced by at least 80% with fresh medium, thereby generating a plurality of second cell aggregates comprising HECs. In a further embodiment, step (b1) lasts for 2 days. In a further embodiment, step (b2) lasts for 4 days. In a further embodiment, the culture medium is renewed for at least 80% every day during step (b2), and VEGF, bFGF, CHIR99021 and SB431542 are administered to the cell culture as follows: day 1-VEGF, bFGF, CHIR99021 are added to the culture medium (thus removing the BMP4-containing medium from the cell culture), day 2-VEGF, bFGF, CHIR99021 and SB431542 are added to the culture medium, day 3-VEGF, bFGF and SB431542 are added to the culture medium, and day 4-VEGF and bFGF are added to the culture medium. In a further embodiment, during step (b), VEGF, bFGF and BMP4 are administered at 25 ng / mL and CHIR99021 and SB431542 are administered at 3 μM in the cell culture. In a further embodiment, the second cell aggregates resulting from step (b) are subjected to step (c) as described herein to obtain HSCs, in particular using a component-defined HSC differentiation medium as described herein. It is understood that the second cell aggregates are advanced to step (c) without disruption of the second cell aggregates, e.g. to perform a cell selection or cell separation step.

[0094] In an embodiment, the method of the present invention further comprises a step (c) of culturing a plurality of second cell aggregates in a culture medium for inducing differentiation of HECs contained in the plurality of second cell aggregates to generate a plurality of third cell aggregates that generate hematopoietic stem cells (HSCs), and allowing the HSCs to be released from the plurality of third cell aggregates in a culture medium for obtaining a population of human HSCs. It is understood that the second cell aggregates are advanced to step (c) without disruption of the second cell aggregates to perform a cell selection or separation step. The second cell aggregates remain intact and are not subjected to any type of intended cell dispersion methodology (whether physical disruption, enzymatic or otherwise chemical) to at least partially disrupt the second cell aggregates.

[0095] In step (c), the second cell aggregates are cultured in a culture medium for inducing differentiation of HECs contained in the second cell aggregates to generate a third cell aggregate that generates hematopoietic stem cells (HSCs), and the third cell aggregates are allowed to release HSCs in a culture medium for obtaining a population of human HSCs, thereby forming a single cell population containing HSCs in suspension in the culture medium. During step (c), the HECs in the second cell aggregates differentiate to become at least a portion of HSCs. It is understood and preferred that the HSCs are released from the aggregates via budding. Budding is a process for releasing cells from a dense population of cells, such as a cluster of cells, such as a cell aggregate, that is well described in the art.

[0096] In some embodiments, the method according to the present invention does not include a cell selection or cell separation step, especially prior to the formation of HSCs. Preferably, the single cells or single HSCs are derived only from cell aggregates that are released (e.g., via sprouting) from the cell aggregates. This means that no mechanical or other force is applied to disperse the cells present in the first, second or third cell aggregates described herein. For example, no (sub)population of cells present in the culture medium is specified that is selectively collected, removed or separated from other cells present in the culture medium or culture vessel based on their marker profile.

[0097] In some embodiments, step (c) comprises steps (c1) and (c2), and step (c2) comprises separating a single cell population comprising HSCs (spontaneously budded from the third cell aggregate) from the remaining cell aggregates. It is contemplated that the HSCs are separated from the remaining cell aggregates by using methods commonly used in the art, such as, but not limited to, filtration, centrifugation or elutriation (e.g., counterflow centrifugal elutriation), for example, by using a Gibco™ CTS™ Rotea™ counterflow centrifugation system. In one preferred embodiment, separation is by counterflow centrifugal elutriation. Preferably, step (c1) comprises culturing a plurality of second cell aggregates in a culture medium for inducing differentiation of HECs contained in the plurality of second cell aggregates to generate a plurality of third cell aggregates that generate hematopoietic stem cells (HSCs), and allowing HSCs to be released from the plurality of third cell aggregates in a culture medium for obtaining a population of HSCs, thereby forming a single cell population comprising HSCs in suspension in the culture medium.

[0098] Suitable culture conditions for differentiating HECs into HSCs are known to those skilled in the art. For example, IMDM medium can be used. The culture medium can be optionally supplemented with additives selected from, but not limited to, BSA (e.g., 0.04% to 1%, preferably about 0.2%), ITS-X (e.g., 0.2% to 5%, preferably about 1%), β-me (e.g., 11 to 275 μM, preferably about 55 μM), ascorbic acid-2P (e.g., 10 to 250 μg / mL, preferably about 50 μg / mL), and GlutaMax (e.g., 0.2% to 5%, preferably about 1%). Growth factors for inducing and / or expanding HSCs for use in the methods of the present invention can be selected from the group including TPO (e.g., 10-250 ng / mL, preferably about 50 ng / mL), hSCF (e.g., 10-250 ng / mL, preferably about 50 ng / mL), FIT3-L (e.g., 10-250 ng / mL, preferably about 50 ng / mL), IL-6 (e.g., 2-50 ng / mL, preferably about 10 ng / mL), and / or IL-3 (e.g., 2-50 ng / mL, preferably about 10 ng / mL). Non-limiting examples of other suitable growth factors for step (c) are Wnt3a (e.g., 1-500 ng / mL), bFGF (e.g., 1-100 ng / mL, preferably about 10 ng / mL), FICZ (e.g., 0.5-20 μM) or TCDD (e.g., 1 nM-1 μM). Other suitable growth media for step (c) include, but are not limited to, StemPro, Hams-F12, APEL and RPMI. Suitable supplements include, but are not limited to, KO-SR, N-2 supplement, B-27 supplement.

[0099] In some embodiments, the method according to the invention comprises that the culture medium for inducing differentiation of HEC comprises bFGF. The culture medium for inducing differentiation of HEC preferably comprises bFGF in combination with one or more additional growth factors that initiate HSC differentiation and / or expansion. For example, the culture medium may comprise bFGF in combination with FLT3L and / or SCF and one or more additional growth factors that initiate HSC differentiation and / or expansion. bFGF is added to the HSC differentiation medium at a concentration of 5-125 ng / mL, 5-75 ng / mL, 5-50 ng / mL. In certain embodiments, bFGF is present in the HSC differentiation medium at about 25 ng / mL or about 10 ng / mL.

[0100] In one preferred embodiment, the method according to the invention comprises that the culture medium for inducing differentiation of HEC comprises bFGF in combination with one or more additional growth factors that initiate HSC differentiation and / or expansion, e.g., in combination with FLT3L and one or more additional growth factors that initiate HSC differentiation and / or expansion, and / or in combination with, e.g., SCF and one or more additional growth factors that initiate HSC differentiation and / or expansion.

[0101] It is contemplated that the one or more additional growth factors that initiate HSC differentiation do not include one or more notch ligands. Thus, in one embodiment, the method according to the present invention, preferably step (c) in which a culture medium is provided to induce differentiation of HECs, does not include providing a notch ligand to the culture medium and / or cells. More preferably, the HECs and / or HSCs obtained herein are not exposed to or contacted with a notch ligand, e.g., Jagged1, Jagged2, Delta-like 1 (DLL1), Delta-like 3 (DLL3), or Delta-like 4 (DLL4), during the method of the present invention.

[0102] In some embodiments, when provided in the culture medium for differentiating HECs into HSCs in step (c) of the method of the invention, IL-3 can be provided at a concentration of 0.5 ng / mL to 50 ng / mL. In a particular embodiment, IL-3 is present in the HSC differentiation medium at 1 ng / mL, in particular to provide HSCs for further differentiation into lymphoid cells, such as NK cells or T cells (including Treg cells). In an alternative particular embodiment, IL-3 is present in the HSC differentiation medium at 10 ng / mL, in particular to provide HSCs for further differentiation into myeloid cells.

[0103] In certain embodiments, in step (c), e.g., in step (c1), the culture medium for inducing differentiation of HECs comprises the growth factors TPO, hSCF, Flt3-L, IL-6 and IL-3. In further embodiments, said hSCF, TPO and Flt3L are present at about 50 ng / mL, and IL-3 and IL-6 are present at about 10 ng / mL.

[0104] In certain embodiments, e.g., in step (c), e.g., in step (c1), the culture medium for inducing differentiation of HECs comprises the growth factors hSCF, Flt3-L and bFGF. In further embodiments, the hSCF and Flt3L are present at about 50 ng / mL and bFGF is present at about 10 ng / mL.

[0105] In another particular embodiment, in step (c), e.g., in step (c1), the culture medium for inducing differentiation of HECs comprises the growth factors TPO, hSCF, Flt3-L, IL-6, IL-3 and bFGF. In a further embodiment, the hSCF, TPO and Flt3L are present at about 50 ng / mL, bFGF and IL-6 are present at about 10 ng / mL, and IL-3 is present at a concentration of 1-10 ng / mL, including between 1 and 10 ng / mL.

[0106] The specific culture medium compositions for HEC and / or HSC differentiation described herein are also considered part of the present invention.

[0107] Alternatively, instead of using a culture medium for HSC differentiation in step (c), it is further envisaged that the medium used in step (c) can be a medium for further differentiation towards hematopoietic lineages. A non-limiting example is differentiation towards lymphoid or myeloid cell lineages. Without wishing to be bound by theory, the inventors believe that efficient differentiation can be achieved, for example, by directly differentiating aggregates containing HECs towards lymphoid or myeloid lineages. It is further theorized that differentiation through HSCs still occurs when HECs are cultured in a lineage-specific culture medium (e.g., lymphoid or myeloid lineage-specific culture medium), meaning that the aggregates still generate HSCs, which are released from the aggregates to further differentiate into lymphoid or myeloid cells. This is further supported by the data presented in Figures 2C-2F, in which HECs were directly differentiated into T cells (lymphoid; Figures 2C-2E) or monocytes (myeloid; Figure 2F). Lymphoid cells can be seen to express HEC / HSC markers (CD34, FIG. 2C) at day 11, T cell precursor markers (CD1a, FIG. 2D) at day 14, and T cell markers (CD3 and CD4, FIG. 2E) at day 32. Myeloid cells can be seen to express HSC markers (CD45) and monocyte markers (CD14, CD1b; FIG. 2F) at day 28.

[0108] Thus, in embodiment step (c), a plurality of second cell aggregates are cultured in a culture medium for inducing differentiation of HECs contained in the plurality of second cell aggregates to generate a plurality of third cell aggregates that generate hematopoietic stem cells (HSCs), and the HSCs are allowed to be released from the plurality of third cell aggregates in a culture medium for obtaining a population of human HSCs, where the culture medium in step (c) is for differentiation into lymphoid or myeloid lineage cells. Culture conditions that result in either lymphoid or myeloid lineage differentiation are known to those skilled in the art. For example, for myeloid lineage differentiation, exemplary differentiation media that can be used from day 6 onwards are as follows: Medium 1: XVIVO15+MCSF 100ng / mL+IL3 25ng / mL; or Medium 2: XVIVO15+TPO 50ng / mL+Flt3L 10ng / mL+huSCF 50ng / mL+MCSF 80ng / mL+GMCSF 10ng / mL. For lymphoid / T cell differentiation, an exemplary differentiation medium that can be used starting from day 6 onwards is as follows: HEC aggregates plated on vitronectin coated tissue culture plastic in XVIVO15 medium + VEGF 50 ng / mL + huSCF 100 ng / mL + bFGF 10 ng / mL + IL7 20 ng / mL).

[0109] Thus, in step (c), the focus can be on HSC differentiation to maximize the differentiation and expansion of HSCs that can either be used directly or cryopreserved for subsequent use and differentiation into the desired cell type. Alternatively, HECs can be immediately pushed to differentiate directly towards a specific lineage (e.g., lymphoid or myeloid), thereby reducing process steps, simplifying the process, and reducing the overall length of the manufacturing process.

[0110] In an embodiment, step (c) is carried out for at least about 4 days, such as for example at least 3, 4, 5, 6, 7 or 8 days, and / or up to about 14 days, such as for example up to 14, 13, 12, 11, 10, 9 or 8 days. Preferably, step (c) is carried out for 3 to 14 days, such as for example 4 to 12, 5 to 10, or 6 to 8 days. Alternatively, step (c) is carried out for 5 to 14 days, 8 to 14, 10 to 14, or 12 to 14 days. It has surprisingly been found that using the method of the invention it is possible to allow the aggregates to generate HSCs for an extended period of time. The HSCs so obtained can be further expanded, for example after cryopreservation and thawing of the cells, for example as discussed herein. The cells can be expanded in the same or a different (bio)reactor.

[0111] In an embodiment, at least 60-90% (eg, at least 60, at least 70, at least 80, or at least 90%) of the cells released from the plurality of third cell aggregates are HSCs.

[0112] In some embodiments, the single cell population separated from the remaining cell aggregates comprising HSCs comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80% HSC cells, or at least 90% HSCs, where the HSC cells express the markers CD34, CD43 and / or CD45.

[0113] In embodiments, the culture medium during step (c) is replaced every day, every other day, every 3 days, every 4 days, or a combination thereof (e.g., the first replacement is after 2 days and the subsequent replacement of the culture medium is after 4 days). In certain embodiments, the culture medium is renewed more frequently at the beginning of step (c). In certain preferred embodiments, the culture medium during step (c) is replaced or renewed daily.

[0114] In some embodiments, during step (c), all the medium is replaced or renewed. However, it has been surprisingly found that it is possible to design a robust and reproducible manufacturing process that results in a large amount of well-differentiated cells in a closed culture system, not by withdrawing the entire culture medium from the culture vessel and replacing it with fresh culture medium, but by withdrawing only a portion of the culture medium from the culture vessel before the subsequent fresh culture medium (and, if necessary, growth factors, such as those disclosed herein) are added to the remaining culture medium in the culture vessel (and thus a portion of the previous medium is mixed with the subsequent medium). For example, in one embodiment, up to 95 vol.%, 90 vol.%, 85 vol.%, or 80 vol.% of the culture medium in the culture vessel is withdrawn, thereby allowing an efficient switch of medium composition and / or the recovery of (single) cells, such as HSC cells, or substances secreted by the cells into the culture medium. For example, in this embodiment, if the culture vessel contains 10 liters of culture medium, then up to 9500, 9000, 8500 or 8000 milliliters of culture medium may be withdrawn from the culture vessel (and from which HSCs may be isolated and obtained).

[0115] At the same time, it has been found that preferably at least 30 vol.%, 40 vol.%, 50 vol.%, 60 vol.% or 70 vol.% of the culture medium is recovered from the culture vessel. Thus, in some embodiments, for example, 30 vol.% to 95 vol.%, or 40 vol.% to 95 vol.%, or 50 to 95 vol.%, or 60 to 90 vol.%, or 60 to 80 vol.%, or 70 to 90 vol.%, or 70 to 80 vol.% of the culture medium is recovered from the culture vessel and preferably replaced or renewed with new culture medium. As will be understood by the skilled artisan, the amount or percentage of medium recovered may vary between different moments of medium recovery according to the method of the present invention. For example, during a first medium withdrawal in step (c), 70 vol.% of the culture medium in the culture vessel can be withdrawn and replaced, and during a subsequent medium withdrawal in step (c), the same, more or less medium (e.g., 50 vol.% or 75 vol.%) can be withdrawn and replaced. In some embodiments, growth factors are added and provided directly to the culture medium already present in the culture vessel or closed culture system, without substantial replacement of old culture medium or substantial addition of new culture medium.

[0116] The markers used or described herein are generally known to those skilled in the art of stem cell differentiation. For easy reference, the following table is provided: Marker Alias ​​Coding gene (human) CD34 ENSG00000174059 CD73 NT5E ENSG00000135318 CD144 CDH5 ENSG00000179776 CD45 PTPRC ENSG00000081237 OCT4 POU5F1 ENSG00000230336 SOX2 ENSG00000181449 NANOG ENSG00000111704 CD43 SPN ENSG00000197471 CD1a ENSG00000158477 CD7 ENSG00000173762 CD4 ENSG00000010610 CD117 KIT ENSG00000157404 CD49f ITGA6 ENSG00000091409 CD90 THY1 ENSG00000154096 CD44 ENSG00000026508 CD14 ENSG00000170458 CD1b ENSG00000158485 Marker Expression Profile CD34 HECs and HSCs are CD34 positive CD73 HECs are CD73 negative CD144 HECs are CD144 positive CD45 HSCs are CD45 positive OCT4 PSCs are OCT4 positive SOX2 PSCs are SOX2 positive NANOG PSCs are NANOG positive CD43 HSCs are CD43 positive CD1a proT cells (T cell precursors) are CD1a positive CD7 thymocytes / mature T cells are CD7 positive CD4 T helper cells, monocytes, macrophages, and dendritic cells are CD4 positive CD117 HSCs are CD117 positive CD49f LT-HSCs are CD49f positive CD90 HSCs are CD90 positive CD44 T cells are CD44 positive CD14 macrophages are CD14 positive CD1b Dendritic cells, monocytes and thymocytes are CD1b positive

[0117] In embodiments, the plurality of first cell aggregates have an average size of about 20-55 micrometers, about 25-50 micrometers, or about 30-40 micrometers, and the plurality of second cell aggregates have an average size of about 35-200 micrometers in diameter. In further embodiments, the plurality of first cell aggregates have an average size of about 49, 48, 47, 46, 45 micrometers or less in diameter.

[0118] It is understood that while differentiating, the cells in the plurality of first and second cell aggregates also proliferate, meaning that the cell aggregates grow in size over time. Thus, in an embodiment, for example, after 3-8 days of step (a), for example, preferably after 5-6 days of step (a), for example, on day 6, the plurality of second cell aggregates have an average size of about 150-600 micrometers in diameter. For example, the aggregates can have an average size of at least 150, 160, 170, 180, 190, or even 200 micrometers, and / or an average size of 600, 580, 560, 540, 520, or 500 micrometers in diameter or less. Thus, the average size of the plurality of second cell aggregates is preferably increased by 10-20 times, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times, when compared to the average size of the plurality of first cell aggregates.

[0119] In some embodiments, the plurality of second cell aggregates have an average size of about 150-600 μm in diameter.

[0120] In an embodiment, the HECs contained in the plurality of second cell aggregates express the markers CD34 and / or CD144; and / or the plurality of second cell aggregates comprises at least 30% cells expressing CD34; at least 30% cells expressing CD144; and / or up to 30% cells expressing CD73. It is further theorized that the absence of the marker CD235a can be used, and thus, in an alternative embodiment, the HECs contained in the plurality of second cell aggregates express the markers CD34 and / or CD144; and / or the plurality of second cell aggregates comprises at least 30% cells expressing CD34; at least 30% cells expressing CD144; and / or up to 30% cells expressing CD73; and / or preferably up to 30% cells expressing CD235a. Even when a cell is considered not to express a marker, it is believed that the marker may still be present on the cell in small amounts. Thus, when referring to a marker, the term "expressed" or "not expressed" refers to whether the marker is present above or below a certain threshold. The threshold for each marker may be easily determined by the skilled artisan by calibrating with a reference sample, as the skilled artisan knows and understands. Preferably, the cells contained in the plurality of second cell aggregates include at least 30% cells expressing CD34, e.g., 30%, 35%, 40%, 45%, 50% or more cells expressing CD34, and / or at least 30% cells expressing CD144, e.g., 30%, 35%, 40%, 45%, 50% or more cells expressing CD144, and / or up to 30% cells expressing CD73, e.g., 25%, 20%, 15%, 10%, 5%, 2% or less cells expressing CD73. Alternatively, the cells contained in the plurality of second cell aggregates contain up to 30% cells expressing CD235a, for example 25%, 20%, 15%, 10%, 5%, 2% or less cells expressing CD235a.

[0121] In an embodiment, the human HSCs released from the plurality of third cell aggregates, preferably in the form of a single cell suspension, express the markers CD34, CD43 and / or CD45. Preferably, the population of human HSCs obtained in step (c) (or in steps (c1) and / or (c2)) comprises at least 50% cells expressing CD34; at least 50% cells expressing CD43; at least 50% cells expressing CD45; and / or up to 30% cells expressing CD14. Preferably, at least 50% or more of the cells express the markers CD34, CD43 and CD45. Preferably, the human HSCs in the form of a single cell suspension and / or the population of human HSCs obtained in step (c) comprise at least 50% cells expressing CD34, such as 50%, 55%, 60%, 65%, 70%, 75% or more cells expressing CD34, and / or comprise at least 50% cells expressing CD43, such as 50%, 55%, 60%, 65%, 70%, 75% or more cells expressing CD43, and / or comprise at least 50% cells expressing CD45, such as 50%, 55%, 60%, 65%, 70%, 75% or more cells expressing CD45. Preferably, the human HSCs in the form of a single cell suspension and / or the population of human HSCs obtained in step (c) comprises up to 30% cells expressing CD14, for example 30%, 25%, 20%, 15%, 10% or less cells expressing CD14.

[0122] In embodiments, the population of human HSCs is in the form of a single cell suspension comprising at least 50% HSCs, or at least 75% HSCs, or at least 90% HSCs, such as, for example, 50%, 60%, 70%, 80%, 90% or more HSC cells, preferably wherein the HSC cells express the markers CD34, CD43 and / or CD45.

[0123] In an embodiment, a step of culturing a plurality of first cell aggregates in step (b), wherein step (b) includes a step (b1) of culturing in which the culture medium contains a SMAD pathway agonist, preferably BMP4, and a step (b2) of culturing in which the culture medium is at least partially replaced with fresh medium not containing the SMAD pathway agonist, and step (b1) is prior to step (b2).

[0124] In an embodiment, the human HSCs released from the plurality of third cell aggregates are cryopreserved to obtain cryopreserved HSCs.Those skilled in the art will be aware of suitable protocols for cryopreservation.For example, Cryostor® CS10 Cryomed™ medium can be used.

[0125] In an embodiment, the method includes a step (d) which comprises further culturing the human HSCs and / or cryopreserved HSCs in a culture medium to induce their proliferation. The HSCs can be isolated from the culture and either used directly or cryopreserved. For example, the HSCs can be matured in a suitable medium. Suitable media are known to those skilled in the art, and for example, IMDM medium can be used, which may contain, for example, but is not limited to, BSA (e.g., 0.04% to 1%, preferably about 0.2%), ITS-X (e.g., 0.2% to 5%, preferably about 1%), β-me (e.g., 11 to 275 μM, preferably about 55 μM), ascorbic acid-2P (e.g., 10 to 50 μg / mL, preferably about 50 μg / mL), GlutaMax (e.g., 0.2% to 5%, preferably about 1%), TPO (e.g., 20 to 500 ng ... 100-2500 nM, preferably about 500 nM-1000 nM), and / or Scriptaid (e.g., 0.2-5 μM, preferably about 1 μM). It has also been found that a medium containing UM729, preferably at the concentrations as provided herein, and preferably a medium for maturing and / or expanding HSCs obtained by the method of the present invention, makes it possible to obtain matured and / or expanded HSCs of high quality, i.e. maintaining their biomarker profile as present in the cell population resulting from step (c) as described herein.

[0126] In particular, where downstream differentiation of HSCs towards myeloid lineage is envisaged, it is contemplated that further addition of IL-3 will further increase the yield / expansion ratio of HSCs obtained by the methods described herein.

[0127] In an embodiment, the expansion ratio PSC:HSC is at least 50, at least 100, or at least 400, for example after expansion of HSC obtained using the method of the present invention. For example, during steps (a) and (b) of the method, the expansion ratio PSC:HSC is at least 10, or even 20, 30, 40, 50, 60, 70, or 80 or more. Additionally, in optional step (d) of the method, the expansion of HSC is preferably at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. As used herein, expansion of HSC means (number of HSC at the end of step (d)):(number of HSC at the start of step (d)). Thus, in an embodiment, the expansion of HSC in step (d) is 2 or more, preferably 4 or more, and most preferably 10 or more. As used herein, the expansion ratio PSC:HSC refers to the amount of cells (in this case HSC) generated compared to the amount of cells (in this case PSC) provided in the culture in step (a). For example, if 10.000 PSCs are provided and 550.000 HSCs are generated / obtained, the expansion ratio is greater than 50, i.e. 55.

[0128] It is understood that UM729 is a pyrimidoindole derivative, a compound previously described as an agonist of hematopoietic stem cell self-renewal (Fares et al., 2014 Science 345, 1509-1512). Another agonist with similar action is UM171.

[0129] In embodiments, the human HSCs are further cultured to induce differentiation of the cells into common erythroid / megakaryocyte progenitors, erythrocytes, megakaryocytes, platelets, common lymphoid progenitors, lymphoid lineage cells, lymphocytes, T lymphocytes, natural killer (NK) cells, common myeloid progenitors, myeloid-derived suppressor cells, common granulocyte-monocytic progenitors, monocytes, macrophages, and / or dendritic cells. Non-limiting examples of T lymphocytes are helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Regulatory T cells may also be referred to as Treg cells, and may be resting Treg cells, activated Treg cells, or immunosuppressive Treg cells. Induction of these cells can be performed using methods known to those skilled in the art. In certain embodiments, the HECs and / or HSCs are further cultured to induce differentiation of the cells into lymphoid lineage cells, e.g., lymphoid progenitor cells, T cells (including α / β T cells and γ / δ T cells), helper T (TH) cells, regulatory T (Treg) cells, natural killer (NK) cells. In alternative embodiments, the HECs and / or HSCs are further cultured to induce differentiation of the cells into myeloid lineage cells. Alternatively, the HECs and / or HSCs are further cultured to induce differentiation of the cells into myeloid lineage cells.

[0130] In an embodiment, the cell culture is carried out in a bioreactor.

[0131] There are many types of closed culture systems, culture vessels or bioreactors that are suitable for the methods as disclosed herein. In particular, the bioreactors as provided herein preferably include a stirring mechanism or other means that allow mixing of the culture medium containing the cells during cultivation. Examples of suitable systems are custom stainless steel / glass reactors or single-use systems such as Ambr and Biostat (Sartorius), PBS (PBS biotech), DASbox and Bioflo (Eppendorf), Appiflex (Applikon), Wave and Xuri (GE / Cytiva), with typical volume ranges from a few milliliters to hundreds of liters. Further specific examples of closed culture systems for carrying out the methods provided herein are described in WO2022019768.

[0132] Additionally, the bioreactor as disclosed herein can be used with oxygen and CO 2 The culture vessel may include a control device for the control of the cell density, the pH value of the culture medium, the lactate concentration, and / or a probe for measuring the amount of dissolved oxygen contained in the culture medium and introduced into the culture vessel. Such probes and control devices are known to those skilled in the art.

[0133] When the method is preferably carried out in a closed system or (closed) bioreactor, it is preferred that the method does not include a step comprising cell selection (i.e. a cell selection step) and / or enrichment for a specific cell type (i.e. a specific cell type enrichment step). In some embodiments, non-single cell sorting is performed on the HECs and / or HSCs obtained herein and / or any other of the cells obtained by further culturing and / or differentiating the HSCs.

[0134] An advantage of performing the methods of the invention in such closed systems or (closed) bioreactors, preferably without said cell selection and / or cell type enrichment steps, is that the methods of the invention enable a process that does not require opening the system and / or bioreactor (e.g. by opening a lid or valve) and thereby exposing the cells to atmosphere, contaminants and / or process steps performed by a human, etc. to perform steps to manipulate the cells.

[0135] Another advantage of the method according to the invention, preferably when carried out in a closed system or (closed) bioreactor, is that it allows obtaining cells and / or cell aggregates with high yield (e.g. PSC:HSC expansion ratio of at least 50, at least 100, or at least 400) and / or high purity (e.g. expressing levels of CD144, CD45, CD43 and / or CD34 markers as described herein). It is therefore contemplated that cells obtained by the method as broadly described herein are suitable for therapeutic use, e.g. use in the treatment of diseases and / or disorders, or at least suitable for differentiation into cells for therapeutic use.

[0136] It is a further advantage of the methods provided herein that the resulting compositions comprising desired cells, e.g., cell cultures comprising HEC or HSC of the methods according to the invention, e.g., cell cultures of PSC, HEC and / or HSC, do not contain (exogenous) feeder cells and / or solid supports (e.g., in the form of nanobeads). Such feeder cells and / or solid supports are well known by those skilled in the art and are commonly used in cell culture processes. When considering therapeutic use of cells produced using the methods described herein, the absence of feeder cells and / or solid supports, e.g., but not limited to, microbeads, is considered advantageous due to the avoidance of potential sources of contamination of the final therapeutic product.

[0137] In a second aspect, the present invention relates to a cell or a composition (of cells) comprising a first population and a second population, wherein the first population is in the form of cell aggregates comprising HEC cells and the second population is in the form of a suspension of single cells comprising HSC cells, preferably the second population comprises at least 50% HSC cells, or at least 75% HSC cells, or at least 90% HSC, wherein the HSC cells express the markers CD34, CD43 and / or CD45, or the cells comprise the second population. Thus, the second population comprises at least 50% cells expressing CD34, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more cells expressing CD34, and / or at least 50% cells expressing CD43, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more cells expressing CD43, and / or at least 50% cells expressing CD45, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more cells expressing CD45.

[0138] In some embodiments, there is provided a population of HECs and / or HSCs and / or differentiated cells selected from the group consisting of lymphoid cells, T cells (including α / β T cells and γ / δ T cells), helper T (TH) cells, regulatory T (Treg) cells and natural killer (NK) cells, obtainable by a method as provided herein.

[0139] In an embodiment, the first, second, and / or third culture medium is at least 1 liter, more preferably at least 10 liters, more preferably at least 50L, more preferably at least 100L in volume.

[0140] In a third aspect, the present invention relates to a population of human HSCs or cryopreserved HSCs as defined herein.

[0141] In an embodiment, the population of HSCs and / or human cryopreserved HSCs comprises at least 70% cells expressing CD34; at least 70% cells expressing CD43; and / or at least 70% cells expressing CD45, and the percentage of cells expressing CD34, CD43 and CD45 remains stable when the cells are expanded according to step (d) for 1 or 2 days in a suitable culture medium at a density of 100 billion to 1 trillion cells per mL, more preferentially 333,333 viable cells / mL. For the purposes of the present invention, the percentage of cells expressing CD34, CD43 and CD45 is considered stable when the percentage of cells is reduced by at most 15%. For example, IMDM medium can be used, and IMDM medium can contain, for example, but is not limited to, BSA (e.g., 0.04% to 1%, preferably about 0.2%), ITS-X (e.g., 0.2% to 5%, preferably about 1%), β-me (e.g., 11 to 275 μM, preferably about 55 μM), ascorbic acid-2P (e.g., 10 to 50 μg / mL, preferably about 50 μg / mL), GlutaMax (e.g., 0.2% to 5%, preferably about 1%), TPO (e.g., 20 to 500 ng / mL, preferably about 100 ng ... may be added, for example, 100 to 500 nM, preferably about 100 ng / mL), hSCF (e.g., 10 to 250 ng / mL, preferably about 50 ng / mL), FIT3-L (e.g., 10 to 250 ng / mL, preferably about 50 ng / mL), IL-6 (e.g., 2 to 50 ng / mL, preferably about 10 ng / mL), IL-3 (e.g., 2 to 50 ng / mL), UM729 (e.g., 100 to 2500 nM, preferably about 500 nM), and / or Scriptaid (e.g., 0.2 to 5 μM, preferably about 1 μM).

[0142] For example, the population of cells comprises at least 70%, e.g., 70%, 75%, 80%, 85%, or 90% cells that express CD34, and / or the population of cells comprises at least 70%, e.g., 70%, 75%, 80%, 85%, or 90% cells that express CD43, and / or the population of cells comprises at least 70%, e.g., 70%, 75%, 80%, 85%, or 90% cells that express CD45.

[0143] In a fourth aspect, the present invention relates to a culture medium or a bioreactor comprising a culture medium comprising a cell or a population of cells as described herein or comprising cells or aggregates as defined herein.

[0144] In some embodiments, the present invention relates to a culture system or bioreactor, preferably a closed culture system or a closed bioreactor, comprising a culture medium comprising a cell or population of cells as provided herein or comprising a cell or cell aggregates as provided herein.

[0145] The foregoing description of specific embodiments will clarify the general nature of the invention sufficiently fully that others, by applying knowledge within the skill of the art (including the contents of the references cited herein), may, without undue experimentation, readily modify and / or adapt such specific embodiments to various applications without departing from the general concept of the invention. Thus, such adaptations and modifications are intended to be within the meaning and equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[0146] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are fully incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also fully incorporated by reference.

[0147] It is to be understood that the phrases or terms used herein are for purposes of description and not of limitation, and thus should be interpreted by one of ordinary skill in the art in light of the teachings and guidance presented herein, in combination with the knowledge of those skilled in the art.

[0148] It will be understood that all details, embodiments and preferences discussed with respect to one aspect of an embodiment of the invention are equally applicable to any other aspect or embodiment of the invention, and therefore it is not necessary to detail all such details, embodiments and preferences separately with respect to every aspect.

[0149] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided for purposes of illustration and are not intended to be limiting of the invention. Further aspects and embodiments will be apparent to those skilled in the art. EXAMPLES

[0150] Working Example Example 1 result Aggregate size during differentiation on days 0–2 After 3 weeks of preculture, PSCs were inoculated into either AggreWell™ 800 plates or a DASbox stirred tank bioreactor system with pitched blades to form aggregates. After 2-3 days of inoculation, AggreWell™ 800 plates produced aggregates with an average diameter of 250 μm, and the bioreactor produced aggregates of 45 μm (Figure 2A). Subsequent parallel culture of aggregates with different sizes showed that smaller aggregates differentiated more efficiently into populations positive for HEC markers. Briefly, 7 days after formation (corresponding to 6 days of culture in HEC differentiation medium), the population was 49.28% positive for CD34, 54.75% positive for CD144, and had low expression (12.12%) for CD73, a marker for HEC precursors (Figure 2B). In contrast, larger aggregates expressed only 11.48% and 11.81% CD34 and CD144, respectively. At day 6, aggregates from this condition had a size of 360±36 μm and a density of 867,683 cells per mL, representing an approximately 40-fold expansion between days 0 and 6.

[0151] When cultured in T-lymphocyte-inducing conditions, the small aggregates proceeded to successfully form a population positive for the hematopoietic cell marker CD45 (23.38%; Fig. 2C), but only 16.76% expressed CD1a, a marker indicative of further differentiation towards the lymphoid lineage. Further culture yielded lymphoid precursors (45.47% CD1a+; Fig. 2D) and subsequently a CD3 / CD4 positive T cell population (48.61%; Fig. 2E), indicating that HECs are capable of forming leukocyte derivatives of lymphoid lineage. In addition, culture of day 7 HECs in conditions suitable for monocytic differentiation gave rise to a population of more than 90% CD14+, CD11b+, or CD45+ (Fig. 2F), demonstrating the ability to form leukocytes of the myeloid lineage.

[0152] Aggregate density at day 0 of differentiation After initial formation, day 0 aggregates were reseeded at different densities to determine optimal conditions for HEC induction. A high seeding density of approximately 33,333 aggregates per mL resulted in the formation of large clusters, likely due to aggregate fusion (Figure 3A). Under these conditions, HEC differentiation was poor, as evidenced by CD34 and CD144 expression at 3.54% and 5.29%, respectively, in day 7 aggregates (Figure 3B). In contrast, a low seeding density of approximately 667 aggregates per mL allowed expression of CD34 at 49.28% and CD144 at 54.75%, with CD73 being expressed in only 12.12% (Figures 2B and 3B). A seeding density of approximately 6,667 aggregates / mL produced intermediate results. Importantly, when cultured in HSC-inducing conditions, a seeding density of 667 aggregates / mL also enabled upregulation of the hematopoietic cell marker CD45 in the highest percentage of the population (69.95%) compared to other conditions tested (43.11% and 7.08% for 6,667 and 33,333 aggregates / mL).

[0153] Medium composition for HSC generation from day 0 to day 6 of differentiation Further refinement of the HSC differentiation process was explored by testing different media formulations between days 0 and 6 of differentiation. Omission of BMP4 supplementation during media changes performed between days 2 and 6 of differentiation generated HSCs with a more stable identity. Specifically, this media formulation reduced the formation of the undesired cell population bearing the primary hematopoietic marker CD235a from approximately 11.2% to 2.06% (Figure 4A) as well as the undesired CD73+ cell population at differentiation day 6 (Figure 4B). At the same time point, BMP4 omission did not affect the efficiency of HEC formation, as evidenced by an unchanged CD34+ / CD144+ / CD7- population (Figure 4B). Changing the BMP4 concentration from 25 ng / mL to 10 ng / mL or 100 ng / mL between days 0 and 6 increased the CD34+ / CD144+ / CD7- population by approximately 10%, but also increased the variability between experiments as well as the presence of the undesirable CD235a population.

[0154] At later differentiation stages and after culture in HSC-inducing medium, BMP4 omission allowed the formation of a cell population that maintained high purity (>80%) of CD45+ / CD43+ / CD34+ markers at least between differentiation days 10 and 14 (Figure 4C). In contrast, treatment with 25 ng / mL BMP4 between days 0 and 6 resulted in a peak in HSC identity at day 10, which declined from 88.95% to 52.85% by day 14. Combined with an overall increase in the CD44high / CD43+ / CD34+ and a decrease in the CD14+ population (Figure 4D), BMP4 omission from days 2 to 6 improved the efficiency and stability of PSC differentiation to HSC. The efficiency and yield of PSC differentiation to HSC was equally high when performed in small-scale 3D suspension cultures with orbital shakers or in large-scale bioreactors (1,172,000 CD45+ / CD43+ / CD34+ cells / mL; Figure 4E), demonstrating the robustness and scalability of the system.

[0155] HSCs were cryopreserved and successfully thawed using CryoStor CS10 freezing medium, maintaining 90% post-thaw viability and >80% CD45+ / CD43+ / CD34+ identity for at least 3 days. Under culture in HSC expansion medium, the number of cells increased approximately 10-fold between days 0 and 7 after thawing (Figure 4F), indicating that the HSCs had a late self-renewal identity.

[0156] Example 2 Effect of first aggregate size method Aggregate size of the first aggregates in D-2~0 vs. D-3~D0 Epithelial cell-derived iPSCs were allowed to form aggregates for either 2 or 3 days prior to the generation of HECs. Cells were seeded in Das Box (DB; 40 M vc / unit). The average size of the first aggregates in diameter for cells allowed to form aggregates for 3 days (D-3 to D0) was approximately 53.5 micrometers (Figure 5A). The average size of the first aggregates in diameter for cells allowed to form aggregates for 2 days (D-2 to D0) was approximately 40.5 micrometers (Figure 5B). After the formation of multiple primary cell aggregates containing PSCs, HE induction (D0-D6) was performed in 6wp suspensions (5000EB / well, in 2 mL medium; 100% renewed). The medium for HE induction followed Feng et al. (WO2020 / 086889A1, referenced herein) with one adaptation, i.e., using 0.33 μM CHIR99021.

[0157] result: Figure 5C shows the measurement of CD144 / CD73 / CD34 markers in HEC obtained from 3 days of DB (40 M vc / unit) aggregate formation (D-3 to D0) and 2 days of DB (DO 15%; 40 M vc / unit) aggregate formation (D-2 to D0). Figure 5B shows that the relatively small average size of the first aggregates (i.e., 40.5 micrometers (D-2 to D0)) improves preferential HEC marker expression CD34 and / or CD144 while simultaneously reducing the marker CD73.

[0158] Example 3 BMP4 Dosing in HEC Differentiation method: Epithelial cell-derived iPSCs were allowed to form aggregates over a period of 2 days according to Example 2. Primary cell aggregates were induced for HEC formation over a period of 6 days (D0-D6) in 6wp suspension (5000EB / well in 2mL medium; 100% refreshed). iPSCs were exposed to different HEC culture medium compositions.

[0159] At D0, the medium composition was IF9S medium containing BMP4 (at concentrations according to Table 1), VEGF at a concentration of 25 ng / mL, bFGF at a concentration of 25 ng / mL.

[0160] On D2, the medium was 100% refreshed and the medium provided to the cells at D2 was IF9S medium containing BMP4, VEGF 25ng / mL, bFGF 25ng / mL and 3μM CHIR99021 at concentrations according to Table 1. On D3, the culture medium was 100% refreshed and the medium provided to the cells was the same as that provided to D2, but further containing 3μM SB431542. On D4, the medium was 100% refreshed and the medium provided to the cells was the same as that provided to D3, but without 3μM CHIR99021. On D5, the medium was 100% refreshed and the medium provided to the cells was the same as that provided to D4, but without 3μM SB431542.

[0161] [Table 1]

[0162] result: The results are shown in Figures 6 to 9. As shown in these figures, conditions containing 25 ng / mL BMP4 between D0 and D2 and 0 ng / mL between D2 and D6 resulted in lower CD235 expression compared to other tested conditions (Figure 6) and good yields (i.e., high fold cell expansion) within 6 days (Figure 7).

[0163] When cells were provided with BMP4 (at either concentration) from day 2 onwards, this was found to correlate with higher CD235 expression (a primary marker) compared to no BMP4 (Figure 6).

[0164] Furthermore, condition 3, which contained a higher BMP4 concentration (100 ng / mL) on both D2-D6 and D3-D7, resulted in poor yields at D6 and a reduction in HSC yields, as evidenced by total HSC density / mL (Figure 8) and marker expression CD45 / CD43 / CD34 (Figure 9)).

[0165] Furthermore, when cells were provided with BMP4 starting on day 2, even at low concentrations (10 ng / mL), expression of myeloid marker (CD14) was observed in HSCs on day 14 (Figure 9), indicating that the cells expanded towards a myeloid cell lineage type of cells (i.e., expressing CD14+ and negative for CD45RA).

[0166] BMP4 at later stages of HE induction of iPSCs (e.g., D2-D6 after HE induction) influences the lineage specificity of the resulting population of HSCs. Thus, by controlling the concentration of BMP4, it is possible to push iPSCs and / or HECs to differentiate directly towards a specific lineage (e.g., lymphoid or myeloid).

[0167] Example 4 Differentiation of HECs to HSCs Epithelial cell-derived iPSCs were allowed to form aggregates for 2 days according to Example 2 and HECs were allowed to form according to Example 3, where the cells were exposed to BMP4 levels according to condition 6 in Table 1.

[0168] Example 4.1 bFGF Various media for HSC generation (D6-D14) in 6wp suspensions (3 mL medium; refreshed 66, 67%) according to Table 2.

[0169] [Table 2]

[0170] On day 14, HSPCs were analyzed for purity and yield.

[0171] result For all four media tested, the average purity of cells expressing the expression markers CD45+ / CD43+ / CD34+ was greater than 80% (FIG. 10A). Medium 2 (i.e., Matsubara-like medium (Matsubara H. et al., "Induction of human pluripotent stem cell-derived natural killer cells for immunotherapy under chemically defined conditions," Biochem Biophys Res Commun. 2019 July 12;515(1):1-8)) resulted in poor yielding HSC cultures compared to medium 1 or medium 3, as expected (Figure 10A). However, medium 4 (Figure 10A) was found to improve the yield (2-3, 6-fold higher than the comparative medium 2 HSC cultures in the respective experiments). Medium 2, 3 and 4 provided high CD7 expression in the CD34+ population of the obtained HSCs, and thus a lymphoid bias. Medium 1 allowed a higher presence of the myeloid marker CD11b (Figure 10B).

[0172] Example 4.2 bFGF&DLL4 Various media for HSC generation (D6-D14) in 6wp suspensions (3 mL media; refreshed 66, 67%) with or without coated DLL4 FC. The conditions tested are shown in Tables 3 & 4:

[0173] [Table 3]

[0174] [Table 4]

[0175] result: The results are shown in FIGS. 11A to 11C and 12A to 12B.

[0176] Example 4.3 IL-3 PSCs were differentiated using a 2 day BMP4 protocol as described for condition 6 in Example 3. HSCs were generated under various conditions using IMDM, 0.2% BSA, 1% ITS-X, 50 μM β-mercaptoethanol, 50 μg / mL AA2P, 2 mM Glutamax, 50 U / mL pen / strep, 50 ng / mL hSCF, 50 ng / mL TPO, 50 ng / mL FLT3l, 10 ng / mL IL-6 (HSC medium control) or IMDM, 0.2% BSA, 1% ITS-X, 50 μM β-mercaptoethanol, 50 μg / mL AA2P, 2 mM Glutamax, 50 U / mL pen / strep, 50 ng / mL hSCF, 50 ng / mL FLT3l (basal medium 2+SCF+FLT3l-condition 10) supplemented with any of the additives mentioned under "HSC generation" in Table 5 (e.g., conditions 2, 3, 12, 13) (Table 5). 5 (see Table 5).

[0177] [Table 5]

[0178] result The results are shown in Figures 13 and 14. In Figure 13, when comparing condition 2 to condition 12, the effect of DLL4 coating is seen in the amount of suspended HSPCs generated (2.5 mln / mL vs 1.8 mln vc / mL). Furthermore, when comparing condition 3 to condition 13, the effect of bFGF addition is seen in an increase in the amount of suspended HSPCs due to the addition of bFGF (1.3 mln / mL vs 1.5 mln vc / mL).

[0179] In Figure 14A, DLL4 coating in condition 12 resulted in lower cell concentrations of cells expressing CD45 / CD43 / CD34 at days 10, 12 and 14 compared to condition 2. Furthermore, the effect of bFGF addition (condition 13) compared to condition 3 is also an increase in cells expressing CD45 / CD43 / CD34 (Figure 14A). This is consistent with the lymphoid expression markers for these conditions in Figure 14B, where the addition of bFGF results in an increase in CD7+ cells (condition 13). Thus, it was found that lowering the concentration of IL-3 (1 ng / mL), i.e. conditions 3 and 13, maximized lymphoid precursor generation (Figure 14B CD7+) and / or minimized myeloid precursor generation (Figure 14C CD11b+).

[0180] Example 4.4 Add after thawing HSC generation (D6-D14) in 6wp suspension (renewal 66,67%) and by differentiation from HEC at D6-D14 using medium containing IMDM, 0.2% BSA, 1% ITS-X, 50 μM β-mercaptoethanol, 50 μg / mL AA2P, 2 mM Glutamax, 50 U / mL pen / strep, 50 ng / mL hSCF, 50 ng / mL TPO, 50 ng / mL FLT3l, 10 ng / mL IL-6 and 10 ng / mL IL-3.

[0181] Thus, HSCs were frozen and thawed using a post-thaw media composition containing IMDM, 0.2% BSA, 1% ITS-X, 50 μM β-mercaptoethanol, 50 μg / mL AA2P, 2 mM Glutamax, 50 U / mL pen / strep, 50 ng / mL hSCF, 50 ng / mL TPO, 50 ng / mL FLT31, 10 ng / mL IL-6, and optionally supplemented with 10 ng / mL IL-3, 1 ng / mL IL-3, and / or 1 μM UM729. Supplementation of the post-thaw media composition with IL-3 and / or UM729 resulted in the specific conditions shown in Table 6.

[0182] [Table 6]

[0183] The readout for this experiment is as follows: Facs on days 0, 3 and 7: 3 panels to measure purity (CD45 / CD43 / CD34, CD7, CD11b). · Growth from day 0 to day 7 to measure yield / quantity.

[0184] result As shown in Figure 15, the addition of UM729 1 μM (condition 7) allows for a higher expansion of HSCs (CD45+ / CD43+ / CD34+) over time post-thaw. Furthermore, Figure 16 shows that the overall post-thaw HSC profile is maintained (see day 3 data) and a higher purity of HSCs (CD45+ / CD43+ / CD34+ in %) is observed with the addition of UM729 1 μM at days 3 and 7 (e.g., when comparing conditions 2 vs 7, 5 vs 8, 6 vs 9), and overall, the addition of UM729 better sustains the HSC marker profile over time (see day 3-7 data for conditions 7, 8 & 9). For example, it is further shown that the highest purity is maintained when combining the addition of UM729 1 μM (conditions 8 and 9) with low IL-3 (e.g., 1 ng / mL) or no IL-3 on days 3 and 7 compared to condition 7 (10 ng / mL IL-3). However, as can be seen in Figure 15, there is an impact of reducing IL-3 concentration on overall yield. In Figure 17, CD7+ (lymphoid lineage marker) maintains stable expression on days 3 and 7 for conditions 8 and 9 (none or low IL-3 + UM729 1 μM).

[0185] In Figure 18, low CD11b (i.e., myeloid lineage marker) is seen at day 7 for both conditions 8 and 9 (none or low IL-3 + UM729 1 μM). Figure 19 shows that the addition of UM729 1 μM (condition 7 (= day 3-7 and day 7-7)) allows for higher generation of CD45+ / CD43+ / CD34+ over time post-thaw. Up to 3 million CD45+ / CD43+ / CD34+ / mL in condition 7 (medium M6) after starting with 250k total cells / mL immediately after thaw.

[0186] Although the invention has now been fully described, it will be understood by those skilled in the art that the same can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

[0187] Reference to known method steps, conventional method steps, known methods, or conventional methods is not an admission that any aspect, illustration, or embodiment of the present invention is in any way disclosed, taught, or suggested in the relevant art.

Claims

1. 1. A method for the in vitro production of a population of hemogenic endothelial cells (HECs), comprising: (a) culturing a suspension of pluripotent stem cells (PSCs), thereby providing a plurality of first cell aggregates comprising said PSCs; (b) culturing the plurality of first cell aggregates in a culture medium for inducing differentiation of the PSCs contained in the plurality of first cell aggregates to generate a plurality of second cell aggregates comprising hemogenic endothelial cells (HECs); Including, - the plurality of first cell aggregates have an average size of less than or equal to about 49 micrometers in diameter; and / or - step (b) comprises a step (b1) of culturing, wherein the culture medium comprises a SMAD pathway agonist, preferably BMP4, and a step (b2) of culturing, wherein the culture medium is at least partially replaced by fresh medium not comprising the SMAD pathway agonist, and step (b1) is prior to step (b2); method.

2. 2. The method of claim 1, wherein the duration of step (b2) is more than 50% of step (b) and / or the duration of step (b1) is less than 50% of step (b), preferably wherein step (b2) lasts for at least 2, 3 or 4 days and / or step (b1) lasts for at most 3, 2 or 1 day.

3. 3. The method of claim 1 or 2, wherein the SMAD pathway agonist is BMP4 and the concentration of BMP4 in the culture medium in step (b1) is between 10 and 40 ng / mL, preferably the (residual) concentration of BMP4 in the culture medium in step (b2) is less than 5 ng / mL.

4. 4. The method according to claim 1, wherein between steps (b1) and (b2) at least 70 vol.%, 80 vol.%, 90 vol.% or 95 vol.% of the culture medium is replaced by fresh culture medium.

5. 5. The method of claim 1, wherein during at least a portion of step (b2), the cells are cultured in the presence of a TGFβ / ALK inhibitor, such as SB431542, and / or a GSK-3β inhibitor, such as CHIR99021.

6. 6. The method according to claim 1, wherein during step (b) the culture medium comprises VEGF and / or bFGF.

7. 7. The method of any one of claims 1 to 6, wherein the plurality of first cell aggregates have an average size of about 20 to 250 micrometers, or about 20 to 150 micrometers, preferably about 20 to 55 micrometers, or about 25 to 50 micrometers, or about 30 to 40 micrometers in diameter.

8. The method of any one of claims 1 to 7, wherein the plurality of second cell aggregates have an average size of about 150 to 600 μm in diameter.

9. The method of any one of claims 1 to 8, further comprising the step (c) of culturing the plurality of second cell aggregates in a culture medium for inducing differentiation of the HECs contained in the plurality of second cell aggregates to generate a plurality of third cell aggregates that generate hematopoietic stem cells (HSCs) in step (c1), and allowing the HSCs to be released (via budding) from the plurality of third cell aggregates in the culture medium for obtaining a population of HSCs, thereby forming a single cell population comprising HSCs in suspension in the culture medium.

10. 10. The method of any one of claims 1 to 9, wherein the plurality of first cell aggregates is provided in a first culture medium at a density of at least 100 aggregates / mL, preferably 100 to 100,000 aggregates / mL.

11. 11. The method of any one of claims 1 to 10, wherein the method does not include a cell selection or cell separation step prior to optionally recovering the single cell population comprising HSCs from the culture medium in step (c).

12. 12. The method of any one of claims 1 to 11, wherein step (c) further comprises a step (c2) in which the single cell population comprising the HSCs is separated from remaining cell aggregates, and wherein step (c1) precedes step (c2).

13. 13. The method of any one of claims 1 to 12, wherein the single cell population comprises at least 50% HSC cells, or at least 75% HSC cells, or at least 90% HSCs, and wherein the HSC cells express the markers CD34, CD43 and / or CD45.

14. 14. The method according to any one of claims 1 to 13, wherein the separation is carried out by countercurrent centrifugal elutriation.

15. 15. The method of any one of claims 1 to 14, wherein the culture medium for inducing differentiation of the HEC comprises one or more growth factors selected from the group consisting of TPO (e.g., 10-250 ng / mL, preferably about 50 ng / mL), hSCF (e.g., 10-250 ng / mL, preferably about 50 ng / mL), FIT3-L (e.g., 10-250 ng / mL, preferably about 50 ng / mL), IL-6 (e.g., 2-50 ng / mL, preferably about 10 ng / mL), IL-3 (e.g., 2-50 ng / mL), Wnt3a, bFGF, FICZ and TCDD.

16. 16. The method of any one of claims 1 to 15, wherein the culture medium for inducing differentiation of the HEC comprises bFGF in combination with one or more further growth factors that initiate HSC differentiation and / or expansion, e.g., in combination with FLT3L and one or more further growth factors that initiate HSC differentiation and / or expansion, and / or in combination with SCF and one or more further growth factors that initiate HSC differentiation and / or expansion.

17. - the HECs contained in the plurality of second cell aggregates express the markers CD34 and / or CD144; and / or - said plurality of second cell aggregates: i. At least 30% of cells expressing CD34; ii. at least 30% of cells expressing CD144; and / or iii. Up to 30% of cells express CD73 Including, 17. The method according to any one of claims 1 to 16.

18. - the HSCs released from the plurality of third cell aggregates (preferably, the HSCs are in the form of a single cell suspension) express markers CD34, CD43 and / or CD45; and / or - said population of HSCs obtained in step (c): i. At least 50% of cells expressing CD34; ii. at least 50% of cells expressing CD43; iii. at least 50% of cells expressing CD45; and / or iv. Up to 30% of cells express CD14 Including, 18. The method according to any one of claims 1 to 17.

19. 19. The method of any one of claims 1 to 18, wherein the HSCs released from the plurality of third cell aggregates are cryopreserved to obtain cryopreserved HSCs.

20. 20. The method according to any one of claims 1 to 19, wherein the method comprises a step (d) comprising further culturing the HSCs and / or the cryopreserved HSCs in a culture medium to induce their proliferation.

21. 21. The method of any one of claims 1 to 20, wherein the expansion ratio PSC:HSC is at least 50, at least 100, or at least 400.

22. 22. The method of any one of claims 1 to 21, wherein the HECs and / or HSCs are further cultured to induce differentiation of the cells into lymphoid cells, such as lymphoid progenitor cells, T cells (including α / β T cells and γ / δ T cells), helper T (TH) cells, regulatory T (Treg) cells, natural killer (NK) cells.

23. 23. The method according to any one of claims 1 to 22, wherein the method is carried out in a closed system or a bioreactor, in particular wherein the method does not comprise a cell selection step or a specific cell type enrichment step.

24. 24. The method according to any one of claims 1 to 23, wherein the cell culture is a suspension culture without a solid support or (exogenous) feeder cells.

25. 1. A composition comprising a first and a second population of cells, wherein the first population of cells is in the form of cell aggregates comprising HEC cells and the second population of cells is in the form of a single cell suspension comprising HSC cells, preferably wherein the second population comprises at least 50% HSC cells, or at least 75% HSC cells, or at least 90% HSC, wherein the HSC cells express the markers CD34, CD43 and / or CD45, or wherein the composition is comprised of the second population of cells.

26. 26. A population of HSCs or cryopreserved HSCs as defined in any one of claims 1 to 25.

27. 25. A population of HECs and / or HSCs and / or differentiated cells selected from the group consisting of lymphoid cells, T cells (including α / β T cells and γ / δ T cells), helper T (TH) cells, regulatory T (Treg) cells and natural killer (NK) cells, obtainable by the method according to any one of claims 1 to 24.

28. A population of HSCs or cryopreserved HSCs, said population of cells comprising: i. At least 70% of cells expressing CD34; ii. at least 70% of cells expressing CD43; and / or iii. At least 70% of cells express CD45 Including, A population of HSCs or cryopreserved HSCs, characterized in that the percentage of cells expressing CD34, CD43 and CD45 remains stable when the cells are expanded for 1 or 2 days according to step (d).

29. 29. A culture system or bioreactor, preferably a closed culture system, comprising a culture medium comprising a cell or a population of cells according to claims 26 to 28 or comprising a cell or cell aggregate as defined in a method according to any one of claims 1 to 24.

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