Compositions and methods for producing megakaryocytes

Abstract

To provide compositions and methods for producing megakaryocytes.SOLUTION: There is provided a method for producing megakaryocyte precursor cells (preMK) and megakaryocytes (MK) from stem cells. The present disclosure further provides compositions comprising preMK and MK and lysates thereof, and also provides methods of using preMK, MK, lysates thereof, and compositions thereof. In one aspect, provided is a method for megakaryocyte production, the method comprising: expanding pluripotent stem cells under low adherent or non-adherent conditions and under agitation, the expanded pluripotent stem cells forming self-aggregating spheroids; differentiating the pluripotent cells in a first culture medium into hemogenic endothelial cells; and differentiating the hemogenic endothelial cells in a second culture medium into megakaryocytic progenitors.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Government support statement This research was supported by the following National Institutes of Health: This invention was supported by grants from the National Institute of Health, Grant Numbers: 1R44HL131050-01, 1R43AI125134-01A1, and 1SB1HL137591-01. The government has certain rights in this invention.

[0002] Related Applications This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 614,117, filed January 5, 2018, which is hereby incorporated by reference in its entirety.

[0003] FIELD OF THE DISCLOSURE The present disclosure relates to megakaryocyte progenitor cells and megakaryocytes, methods of producing compositions of megakaryocyte progenitor cells and megakaryocytes, and methods of use thereof. [Background technology]

[0004] Platelets are blood cells involved in clot formation and vascular repair at sites of active bleeding. Physiologically, platelets are produced in the bone marrow by parent cells called megakaryocytes (MKs), which comprise <0.1% of the cells in the bone marrow. Mature MKs are located outside the sinusoids within the bone marrow, and long structures called proplatelets extend into the circulation. Proplatelets function as an assembly line for platelet production, sequentially releasing platelets from their ends.

[0005] MKs are produced from hematopoietic stem cells in the bone marrow through multiple stepwise differentiation processes. Exposure to various cytokines, chemokines, and growth factors, including thrombopoietin, results in the differentiation of hematopoietic stem cells into multipotent progenitor cells, and then into committed megakaryocytic progenitor cells, also known as pre-MKs. Upon further differentiation, including cell expansion, increased DNA content, endonuclear division, and granule formation, mature MKs are produced. MKs transform their cell mass into elongated platelet precursors to produce / release anucleate platelets.

[0006] Low platelet counts are an important consequence of a variety of diseases and therapies, including cancer treatments, transplants, and surgical procedures, where platelets are an important first-line treatment to prevent death from uncontrolled bleeding. 11 Platelets (units) are derived exclusively from human volunteer donors and must be stored at room temperature to avoid irreversible activation. However, at this temperature, there is a risk of bacterial growth, which limits the shelf life of a platelet unit to 5 days, of which 2 days are consumed for pathogen screening and 1 day for transportation. Therefore, blood centers generally do not have platelet inventories available for transfusion for more than 1.5 days, which rapidly depletes during emergencies. Platelet donor shortages are particularly threatening during crises. Increased demand for general population use alone exceeds supply by approximately 20%, and stockpiles are rapidly depleted during emergencies. Furthermore, wide functional variability between units and donors leads to overtransfusion to ensure effective bleeding control. Therefore, there is an urgent need for megakaryocytes to provide a source of platelets, as well as new and improved methods for generating megakaryocytes. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides methods for producing megakaryocyte progenitor cells (preMKs) and megakaryocytes (MKs) from stem cells. The present disclosure also provides compositions containing preMKs and MKs and their lysates, as well as methods for using preMKs, MKs, their lysates, and the compositions.

[0008] In some embodiments, the present disclosure provides a method for producing megakaryocytes, comprising: expanding pluripotent stem cells under low- or non-adherent conditions and agitation, wherein the expanded pluripotent stem cells form self-aggregating spheroids; differentiating the pluripotent cells into hemogenic endothelial cells in a first culture medium; and differentiating the hemogenic endothelial cells into megakaryocyte progenitor cells in a second culture medium. The step of differentiating the pluripotent cells into hemogenic endothelial cells can be performed on a matrix under adherent conditions. In some embodiments, the step of differentiating the pluripotent cells into hemogenic endothelial cells is performed under low- or non-adherent conditions to allow self-aggregation of the hemogenic endothelial cells.

[0009] In some embodiments, the present disclosure provides a method for producing megakaryocytes, comprising differentiating pluripotent cells into hemogenic endothelial cells in a first culture medium and differentiating the hemogenic endothelial cells into megakaryocyte progenitor cells in a second culture medium, wherein at least one of the differentiating the pluripotent cells and the differentiating the hemogenic endothelial cells is performed on a matrix-coated three-dimensional structure. The three-dimensional structure may be a microcarrier or a microcarrier.

[0010] In some embodiments, the present disclosure provides a method for producing megakaryocytes, comprising differentiating pluripotent cells into hemogenic endothelial cells in a first culture medium and differentiating the hemogenic endothelial cells into megakaryocyte progenitor cells in a second culture medium, wherein at least one of the steps of differentiating the pluripotent cells and differentiating the hemogenic endothelial cells is performed under low-adherence or non-adherence conditions to allow for self-aggregation of the cells.

[0011] In some embodiments, the first culture medium comprises one or more of bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF). The first culture medium may further comprise a WNT modulator. In some embodiments, the second culture medium comprises one or more of stem cell factor (SCF), thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (Flt3-L), interleukin-3 (IL-3), interleukin-6 (IL-6), and heparin.

[0012] In some embodiments, the pluripotent stem cells are human induced pluripotent stem cells.

[0013] In some embodiments, the method may further comprise expanding the pluripotent stem cells in the matrix-coated three-dimensional structure.

[0014] In some embodiments, the method may further include differentiating the megakaryocyte progenitor cells into megakaryocytes in a third culture medium, which may include one or more of stem cell factor (SCF), thrombopoietin (TPO), interleukin-6 (IL-6), interleukin-9 (IL-9), and heparin.

[0015] In some embodiments, the present disclosure provides compositions of megakaryocyte progenitor cells or megakaryocyte progenitor cell lysates produced by the methods of the present disclosure.

[0016] In some embodiments, the present disclosure provides megakaryocytes or megakaryocyte lysates produced by the methods of the present disclosure. In some embodiments, such megakaryocytes express CD42b + , CD61 + , and DNA + is.

[0017] Other features and advantages of the disclosure will become apparent from the following description, and from the claims.

[0018] The present disclosure will be described in the following detailed description with reference to several figures, which are set forth as non-limiting examples of illustrative embodiments, and in which like reference numerals represent like parts throughout the several view figures. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows an overall schematic for scalable differentiation of megakaryocyte progenitor cells (preMKs), megakaryocytes (MKs), and platelets (PLTs) from iPSC lines. [Figure 2] FIG. 2 shows an exemplary directed differentiation protocol from pluripotent stem cells to megakaryocytes in a 2D, matrix-dependent system such as a cell culture plate or flask. [Figure 3] Figures 3A and 3B show the pluripotency of three exemplary clinical-grade hiPSC lines (referred to herein as PBG1, PBG2, and PBG3). Figure 3A shows low-magnification phase-contrast images of PBG1, PBG2, and PBG3 iPSCs forming characteristic growth areas when cultured on a vitronectin matrix using Essential 8 Medium. Figure 3B shows high-magnification images of PBG1, PBG2, and PBG3 iPSCs immunostained for the pluripotency factors Oct4 and Nanog and counterstained with a nuclear dye. [Figure 4]Figures 4A, 4B, 4C, and 4D show cultures of PBG1, PBG2, and PBG3 iPSCs progressing through the phases of directed differentiation from pluripotent stem cells to mature megakaryocytes. Figure 4A shows a schematic of the general timeline of the directed differentiation process. Figure 4B shows actual images of PBG1 cultures during phase 0 (day 0), phase I (days 2 and 5), phase II (days 6+7), and phase III (days +2 and +4). Figure 4C shows actual images of PBG2 cultures during phase 0 (day 0), phase I (days 2 and 5), phase II (days 6+7), and phase III (days +2 and +4). Figure 4D shows actual images of PBG3 cultures during phase 0 (day 0), phase I (days 2 and 5), phase II (days 6+7), and phase III (days +2 and +4). [Figure 5] FIG. 5 is a graph showing the mean CD31+ differentiation efficiency at the end of phase I of directed differentiation of PBG1, PBG2, and PBG3 iPSC lines. [Figure 6A-B]Figures 6A, 6B, and 6C show the purity and yield of megakaryocyte progenitor cells (CD43+CD41+) generated during phase II of directed differentiation of the PBG1, PBG2, and PBG3 iPSC lines. Figure 6A shows representative CD41 / CD43 flow cytometry data for suspension cells recovered from phase II cultures on day 6+6 (from left to right: PBG1, PBG2, PBG3). Figure 6B is a graph showing daily output measurements of CD41+CD43+ (megakaryocyte progenitor) cells released into suspension during representative phase II differentiation cultures of the PBG1, PBG2, and PBG3 iPSC lines. The production of CD41+CD43+ megakaryocytic progenitor cells in PBG1 and PBG2 differentiation cultures was measured up to days 6 + 17, whereas the production of CD41+CD43+ cells in PBG3 differentiation cultures ceased on days 6 + 8. Figure 6C shows the cumulative yield of CD41+CD43+ cells during phase II differentiation culture of clinical-grade hiPSC lines, expressed as the number of CD41+CD43+ cells per well. [Figure 6C] Same as above. [Figure 7] Figures 7A, 7B, and 7C illustrate phase III, the final phase of the directed differentiation protocol that ultimately generates mature megakaryocytes. Figure 7A shows the maturation status of PBG1-, PBG2-, and PBG3-derived cells over time during phase III culture, as measured by the percentage of CD41+ megakaryocyte-lineage cells that also express the mature megakaryocyte marker CD42b. Figures 6B and 6C show light microscopy images of hiPSC-derived megakaryocytes differentiated from PBG1 and PBG2, respectively, on day 4 of phase III. Examples of platelet progenitor outgrowths are indicated by arrows. [Figure 8A-C]Figures 8A, 8B, 8C, 8D, and 8E show the characterization of mature megakaryocytes derived by differentiation of PBG1, PBG2, and PBG3 hiPSCs. Figure 8A shows phase III cells derived from PBG1 hiPSCs, immunostained for β1-tubulin, and nuclei visualized by nucleic acid staining (upper panel). Megakaryocytes derived from PBG1 hiPSCs were also analyzed by electron microscopy (lower panel). Figure 8B shows phase III cells derived from PBG2 hiPSCs, immunostained for β1-tubulin, and nuclei visualized by nucleic acid staining (upper panel). Megakaryocytes derived from PBG2 hiPSCs were also analyzed by electron microscopy (lower panel). Figure 8C shows phase III cells derived from PBG3 hiPSCs. Megakaryocytes derived from PBG3 were immunostained for β1-tubulin, and nuclei were visualized by nucleic acid staining (upper panel). Megakaryocytes derived from PBG3 were not analyzed by electron microscopy (lower panel). Figure 8D shows the percentage of phase III cells derived from PBG1, PBG2, and PBG3 iPSCs that stained positive for intracellular von Willebrand factor. Figure 8E shows the percentage of phase III cells derived from PBG1, PBG2, and PBG3 iPSC lines that stained positive for intracellular platelet factor 4. [Figure 8D-E] Same as above. [Figure 9] Figures 9A, 9B, and 9C show the expansion of pluripotent PBG1 cells on recombinant vitronectin using various growth media. Figure 9A shows the growth of PBG1 in Essential 8 medium. Figure 9B shows the growth of PBG1 in StemFlex medium. Figure 9C shows the growth of PBG1 in Nutristem XF medium. [Figure 10]Figures 10A, 10B, and 10C are graphs showing flow cytometry data assessing the expression of pluripotency markers Tra-1-60, SSEA5, and differentiation marker SSEA1 in PBG1 cells expanded on recombinant vitronectin using various growth media. Figure 10A shows pluripotency marker data from PBG1 cells expanded in Essential 8 medium. Figure 10B shows pluripotency marker data from PBG1 cells expanded in StemFlex medium. Figure 10C shows pluripotency marker data from PBG1 cells expanded in Nutristem XF medium. [Figure 11] Figures 11A, 11B, and 11C show the expansion of PBG1 iPSCs in self-aggregating spheroid cultures in 3D stirred tanks (without matrix). Figure 11A shows microscopic images of PBG1 spheroids over time in culture. Figure 11B shows the increase in cell density over time in PBG1 spheroid cultures. Figure 11C shows the average PBG1 spheroid size over time in 3D cultures. [Figure 12] Figures 12A and 12B show flow cytometry data assessing the expression of pluripotency markers Tra-1-60, SSEA5, and differentiation marker SSEA1 in PBG1 cells expanded in self-aggregating spheroid cultures in 3D stirred vessels (matrix-free). Figure 12A shows pluripotency marker data for PBG1 cells after a single 7-day expansion in 3D stirred vessels. Figure 12B shows pluripotency data for PBG1 cells after four consecutive 6-7 day expansions in 3D stirred vessels. [Figure 13] Figures 13A and 13B show PBG1 iPSCs immunostained for pluripotency factors Oct4 and Nanog and counterstained with a nuclear dye. Figure 13A shows a portion of a 2D colony of PBG-1 iPSCs grown on vitronectin. Figure 13B shows a spheroid of PBG-1 iPSCs grown under 3D agitation conditions (without matrix). [Figure 14]Figure 14 shows karyotype analysis of metaphase chromosome spreads from PBG1 iPSCs grown in 3D stirred tanks for four consecutive 6-7 day expansions, demonstrating a normal karyotype after four rounds of 3D passaging. [Figure 15] FIG. 15 shows the morphological changes that occurred over 6 days of phase I differentiation of PBG1 iPSCs into hemogenic endothelium in a type IV collagen matrix in 2D culture vessels. [Figure 16] Figures 16A and 16B are graphs showing representative phase I differentiation data for PBG1-derived cells. Figure 16A shows a representative flow cytometry analysis of PBG1-derived cells on day 6 of differentiation. Hemogenic endothelial cells are identified by cell surface expression of CD31 and CD34. Figure 16B shows the mean and range of phase I (day 6) differentiation efficiency across 41 independent PBG1-directed differentiations. [Figure 17A] Figures 17A, 17B, and 17C show representative phase II data from PBG1 differentiation cultures. Figure 17A shows a phase II culture at day 6+6, with a hemogenic endothelial (HE) monolayer in the background and megakaryocyte progenitor cells (preMK) released from the monolayer into suspension. Figure 17B shows flow cytometry analysis of phase II suspension cells, identifying CD43+ hematopoietic progenitor cells. Figure 17C shows flow cytometry analysis of CD43+ hematopoietic cells, identifying CD43+CD41+CD14- megakaryocyte progenitor cells (preMK). Contaminating CD43+CD14+ myeloid progenitor cells are also identified in this analysis. [Figure 17B-C] Same as above. [Figure 18]Figures 18A and 18B are graphs showing the average compositional characteristics of phase II suspension cells. Figure 18A shows the average daily purity of released preMK (i.e., %CD41+CD43+CD14- of viable suspension cells) over 10 days of phase II. Figure 18B shows the median, quartiles, and range of contaminating myeloid progenitor cells (i.e., %CD43+CD14+ of viable suspension cells) over 10 days of phase II. All cultures were initiated with PBG1 cells in a type IV collagen matrix in 2D vessels. Data represent 41 independent differentiations. [Figure 19] Figures 19A and 19B are graphs showing the yield of released preMK. Figure 19A shows the average daily yield of released preMK (i.e., viable CD41+CD43+CD14-) per 6-well equivalent (i.e., 2 ml of medium, 9.5 cm2 surface area) during phase II directed differentiation culture initiated with PBG1 iPSCs. Figure 19B shows the cumulative yield of released preMK (i.e., viable CD41+CD43+CD14-) per 6-well equivalent (i.e., 2 ml of medium, 9.5 cm2 surface area) from day 6+4 to day 6+8 of phase II directed differentiation culture initiated with PBG1 iPSCs. Each dot represents an independent PBG1 directed differentiation culture in a type IV collagen matrix in a 2D culture vessel. [Figure 20] Figures 20A, 20B, 20C, and 20D show the differentiation of MKs and the production of platelet precursors in phase III. Figure 20A shows PBG1-derived megakaryocyte progenitor cells on day 1 of phase III (upper panel: high magnification; lower panel: low magnification). Figure 20B shows maturing megakaryocytes on day 2 of phase III (upper panel: high magnification; lower panel: low magnification). Figure 20C shows mature megakaryocytes on day 4 of phase III (upper panel: high magnification; lower panel: low magnification). Figure 20D illustrates spontaneous platelet precursor formation from mature PBG1-derived MKs after 4 days of phase III culture. [Figure 21]Figures 21A, 21B, and 21C show representative flow cytometry analyses from day 3 of a phase III culture initiated from PBG1 iPSCs. Figure 21A identifies the CD61+ (megakaryocytic) fraction of phase III cells. Figure 21B shows flow cytometry analysis of CD61+ megakaryocytic cells, identifying CD42a+CD42b+ mature MKs. Apoptotic CD42a+CD42b- cells can also be identified in this analysis. Figure 21C shows subset breakdown of a representative phase III culture. Non-MKs are CD61-, immature MKs are CD61+CD42a-CD42b-, apoptotic MKs are CD61+CD42a+CD42b-, and mature MKs are CD61+CD42a+CD42b+. [Figure 22] Figures 22A and 22B show the use of laminin 521 and type IV collagen in phase I of directed differentiation of PBG1 cells. Figure 22A shows the progression of phase I differentiation with 4.2 ug / cm2 of human type IV collagen. Figure 22B shows the progression of phase I differentiation with 0.13 ug / cm2 of recombinant human laminin 521. [Figure 23A] Figures 23A, 23B, and 23C show the use of recombinant laminin 521 to support the production and release of megakaryocyte progenitor cells during phase II of directed differentiation of PBG1 cells. Figure 23A shows representative flow cytometry data from phase II of PBG1 differentiation cultures utilizing a support matrix of 4.2 μg / cm2 human type IV collagen. Figure 23B shows representative flow cytometry data from phase II of PBG1 differentiation cultures utilizing a support matrix of 0.13 μg / cm2 recombinant human laminin 521. Figure 23C shows the cumulative yield of released preMKs (i.e., viable CD41+CD43+CD14-) per 6-well equivalent (i.e., 2 ml medium, 9.5 cm2 surface area) between days 6+4 and 6+8 of phase II directed differentiation cultures initiated with PBG1 iPSCs and utilizing support matrices of 4.2 ug / cm2 human type IV collagen or 0.13 ug / cm2 recombinant human laminin 521. [Figure 23B]Same as above. [Figure 23C] Same as above. [Figure 24] Figures 24A and 24B show the production of platelet precursors from MKs differentiated from preMKs generated from laminin-521 culture. A phase III (day 6 + 6 + 3) culture initiated with preMKs from type IV collagen culture is shown in Figure 24A, and a phase III (day 6 + 6 + 3) culture initiated with preMKs from laminin-521 culture is shown in Figure 24B. Red arrows indicate examples of platelet precursors. [Figure 25] Figures 25A and 25B show flow cytometric subset resolution of phase III (day 6 + 6 + 3) cultures. Figure 25A shows flow cytometric subset resolution of phase III (day 6 + 6 + 3) cultures initiated with preMKs from type IV collagen cultures. Figure 25B shows flow cytometric subset resolution of phase III (day 6 + 6 + 3) cultures initiated with preMKs from recombinant laminin-521 cultures. Non-MKs are CD61-, immature MKs are CD61+CD42a-CD42b-, apoptotic MKs are CD61+CD42a+CD42b-, and mature MKs are CD61+CD42a+CD42b+. [Figure 26] Figures 26A, 26B, and 26C show immunofluorescence microscopy images of phase I cultures on laminin-521 at day 6. Figure 26A shows a control culture without WNT agonist. Figure 26B shows a culture in which 0.6 uM of the WNT agonist CHIR98014 was added to the differentiation culture for the first 48 hours of phase I. Figure 26C shows a culture in which 6 uM of the WNT agonist CHIR99021 was added to the differentiation culture for the first 48 hours of phase I. [Figure 27] Figures 27A and 27B show immunofluorescence microscopy images of Phase II cultures on laminin 521 at day 6+4. Figure 27A shows a control culture without WNT agonist. Figure 27B shows a culture in which 0.6 uM of the WNT agonist CHIR98014 was added for the first 48 hours of Phase I. [Figure 28] Figure 28 is a schematic diagram showing an exemplary directed differentiation protocol from PBG1 cells to megakaryocyte progenitor cells using a packed-bed bioreactor strategy, a 3D matrix-dependent method. In the embodiment described herein, laminin-521 coated PTFE Raschig rings are used as macrocarriers to construct the packed-bed. [Figure 29] FIG. 29 shows phase I differentiation of PBG-1 iPSCs on laminin-521 coated Raschig rings on days 3 and 6. [Figure 30] FIG. 30 shows phase II differentiation of PBG-1 iPSCs on laminin-521 coated Raschig rings on days 6+0 and 6+4. [Figure 31] Figures 31A, 31B, and 31C are graphs showing flow cytometry data from phases of PBG1 differentiation proceeding efficiently on Raschig ring supports. Figure 31A shows phase I, day 6, with flow cytometry staining for hemogenic endothelial markers CD31 and CD34. Figure 31B shows phase II, day 6+2, with flow cytometry staining for megakaryocyte progenitor markers CD43 and CD41. Figure 31C shows phase III, day 6+3+3, with flow cytometry staining for CD61 and CD42b. [Figure 32] FIG. 32 is a schematic diagram of an exemplary 3D, matrix-independent method of directed differentiation using self-aggregating iPSC-derived spheroids in a stirred tank. [Figure 33]Figures 33A and 33B show phase 0 and phase I differentiation initiated using self-aggregating spheroids of PBG1 iPSCs. Figure 33A shows a series of photomicrographs of self-aggregated PBG1 iPSC spheroids on day 0, partially differentiated spheroids on day 3, and fully differentiated spheroids containing hemogenic endothelial cells on day 6, starting from single cells dissociated from PBG1 cells on day -1. Figure 33B shows flow cytometry data on day 6 demonstrating successful CD31+CD34+ hemogenic endothelial differentiation using this technique. [Figure 34A-B] Figures 34A, 34B, 34C, and 34D depict phase II of directed differentiation initiated with self-aggregating spheroids of PBG1 iPSCs. Figure 34A shows self-aggregated PBG1-derived spheroids on day 6+4 during phase II of directed differentiation, with preMKs being released from the spheroids into suspension. Figure 34B shows flow cytometry analysis of recovered suspension cells stained for preMK markers CD41 and CD43. Figure 34C shows preMK purity over time during phase II in two different 3D systems: ultra-low attachment vessels on an orbital shaker and spinner flasks. Figure 34D shows preMK yield over time during phase II in two different 3D systems: ultra-low attachment vessels on an orbital shaker and spinner flasks. [Fig. 34C-D] Same as above. [Figure 35A]Figures 35A, 35B, and 35C show phase III MK differentiation from 3D, matrix-independent cultures initiated from self-aggregating spheroids of PBG1 iPSCs. Figure 35A shows a representative flow cytometry analysis from a phase III culture on day 3, identifying the CD61+ (megakaryocyte) fraction of phase III cells, followed by CD42a+CD42b+ mature MKs. Apoptotic CD42a+CD42b- cells can also be identified in this analysis. Figure 35B shows subset decomposition of a representative phase III culture. Non-MKs are CD61-, immature MKs are CD61+CD42a-CD42b-, apoptotic MKs are CD61+CD42a+CD42b-, and mature MKs are CD61+CD42a+CD42b+. FIG. 35C shows how the mature MK fraction within phase III cultures at day 3 compares between 2D (matrix-dependent) and 3D (matrix-independent) approaches. [Fig. 35B-C] Same as above. [Figure 36] 36 shows proplatelet outgrowths of mature MKs harvested from 3D self-aggregating spheroid differentiation cultures. Examples of proplatelet outgrowths are indicated by arrows. [Figure 37] Figure 37 shows PBG1-derived megakaryocytes immunostained for the megakaryocyte-specific protein β1-tubulin. Simultaneously, nuclei were visualized by nucleic acid staining. [Figure 38] Figures 38A, 38B, 38C, 38D, 38E, and 38F show PBG1-derived megakaryocytes immunostained for the alpha granule-specific proteins platelet factor 4 (PF4), von Willebrand factor (VWF), and for the megakaryocyte-specific cell surface marker CD61, and for nuclei. [Figure 39] Figures 39A, 39B, 39C, 39D, 39E, and 39F show PBG1-derived megakaryocytes immunostained for the dense granule-specific proteins LAMP1 and serotonin, and for the megakaryocyte-specific cell surface marker CD61, and for nuclei. [Figure 40]Figures 40A, 40B, 40C, and 40D are electron microscope images showing PBG1-derived megakaryocytes. Figure 40A is an electron microscope image showing PBG1-derived megakaryocytes producing microparticles (see, e.g., arrows). Figure 40B is an electron microscope image showing PBG1-derived megakaryocytes and multivesicular bodies (arrows; enlarged in the inset). Figure 40C is an electron microscope image showing PBG1-derived megakaryocytes characterized by multi-lobed nuclei, glycogen granules, alpha granules, and invaginated membrane systems. Figure 40D is an electron microscope image showing endoplasmic reticulum and mitochondria of PBG1-derived megakaryocytes. [Figure 41A-B] Figures 41A, 41B, and 41C illustrate the characteristic gene expression changes that occur during the directed differentiation of PBG1 cells to megakaryocytes. For all expression analyses, expression in pluripotent PBG1 cells was set to 1, and all other expression values ​​are expressed relative to one another. Figure 41A illustrates the relative gene expression of Oct4, a pluripotency-related gene, in pluripotent PBG1 cells, cells at day 6 (end of phase I), days 6+4 and 6+5 (phase II), and days 6+5+1 to 6+5+4 (phase III). Figure 41B illustrates the relative gene expression of NFE2, a transcription factor crucial for megakaryocyte maturation, in pluripotent PBG1 cells, cells at day 6 (end of phase I), days 6+4 and 6+5 (phase II), and days 6+5+1 to 6+5+4 (phase III). A similar analysis was performed on a panel of relevant genes, and the results of this analysis are summarized in the heatmap shown in Figure 41C, showing that the genes OCT4, SOX2, NANOG, and ZFP42 are downregulated during differentiation, the genes ZFPM1, NFE2, RUNX1, MEIS1, and GATA1 are upregulated during differentiation, and the genes PBX1 and MYC remain at substantially consistent levels. [Figure 41C] Same as above. [Figure 42]Figures 42A, 42B, and 42C present the size distribution of PBG1-derived megakaryocytes. Figure 42A shows a representative example of β1-tubulin staining of PBG1-derived megakaryocytes, which was utilized to collect size measurements of PBG1-MK and compare MK with other sources. Figure 42B shows the size distribution of PBG1-derived megakaryocytes, including the median, quartiles, and range. Figure 42C compares the size distribution data of PBG1-derived MK with megakaryocytes derived from various bone marrow sources. [Figure 43] Figures 43A and 43B show ploidy measurements for PBG1-derived megakaryocytes. Figure 43A shows a representative example of DNA ploidy measurements performed on PBG1-derived megakaryocytes. Figure 43B compares the DNA ploidy measurements of PBG1-MK with MK derived from other sources. [Figure 44] FIG. 44 presents a comparison of the presence or absence and concentration ranges of various factors in hiPSC-MK lysates of megakaryocytes derived by the methods and under certain conditions of the present disclosure. [Figure 45A] Figures 45A, 45B, and 45C show hiPSC platelet production. Figure 45A shows flow cytometry analysis of anucleated and nucleated cells (top, left). Nucleated cells contained numerous CD41+CD42+ megakaryocytes (top, right). Anucleated cells positive for CD41+, CD42+, and calcein AM were assessed using flow cytometry, and platelets were gated by size (1-5 microns). Figure 45B shows an example of platelets recovered from megakaryocyte cultures, assessed by electron microscopy. Figure 45C is a graph showing the cumulative yield of CD41+CD42+Calcein AM+ platelet-sized particles per well during phase III of the directed differentiation protocol described herein. [Figure 45B-C] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0020] While the above-identified figures set forth embodiments of the present disclosure, other embodiments are contemplated as noted in the discussion. The present disclosure presents exemplary embodiments by way of representation and not limitation. Those skilled in the art can devise numerous other modifications and embodiments that fall within the scope and spirit of the principles of the disclosed embodiments.

[0021] The present disclosure is directed to compositions and methods for producing megakaryocyte progenitor cells (preMKs) and megakaryocytes (MKs) from stem cells, such as pluripotent stem cells, e.g., clinical-grade human induced pluripotent stem cells. The methods allow for the continuous production of preMKs from hemogenic endothelial cells over an extended time frame (up to 8 days or longer), which can then be subsequently differentiated into mature MKs. The preMKs and MKs derived by the methods can be distinguished by one or more of the following: size range, ploidy profile, biomarker expression, gene expression, granule composition, and growth factor cytokine and chemokine composition, or a combination thereof. The present disclosure further provides compositions comprising preMKs and MKs and their lysates, as well as methods for using preMKs and MKs and their lysates and compositions.

[0022] definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide those of ordinary skill in the art with general definitions of many of the terms used in this disclosure: Singleton et al. al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994);The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below unless otherwise specified.

[0023] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0024] The term "antibody," as used herein, refers to an immunoglobulin molecule that specifically binds to an antigen. The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic-determining variable regions of the intact antibody.

[0025] By "alteration" or "change" is meant an increase or decrease. The change may be as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or even as much as 40%, 50%, 60%, or even 70%, 75%, 80%, 90%, or 100%.

[0026] "Biological sample" means any tissue, cell, fluid, or other material withdrawn from an organism.

[0027] "Capture reagent" refers to a reagent that specifically binds to a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide.

[0028] By "cell composition" is meant any composition that includes one or more isolated cells.

[0029] "Cell viability" means cell viability.

[0030] As used herein, "clinical grade" refers to cells or cell lines derived or obtained using current Good Manufacturing Practice (GMP) that permit clinical use in humans. GMP is a quality assurance system used in the pharmaceutical industry to ensure that final products meet pre-established specifications. GMP extends to both the manufacturing and testing of final products. GMP requires traceability of raw materials as well as that production be in accordance with validated standard operating procedures (SOPs).

[0031] By "detectable levels" is meant that the amount of analyte is sufficient to be detected using methods routinely used to perform such analyses.

[0032] "Detect" refers to identifying the presence, absence, or amount of the object being detected.

[0033] "Detectable label" refers to a composition that, when attached to a molecule of interest, makes the molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISAs), biotin, digoxigenin, or haptens.

[0034] "Disease" refers to any condition or disorder that damages or interferes with the normal function of cells, tissues, or organs. Examples of diseases include any disease or injury that results in a reduction in cell number or biological function, including ischemic injuries such as stroke, myocardial infarction, or any other ischemic event that causes tissue damage, peripheral vascular disease, wounds, burns, fractures, blunt trauma, arthritis, and inflammatory diseases.

[0035] By "effective amount" is meant the amount of an agent required to produce an intended effect.

[0036] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. The portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. Fragments can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0037] The terms "isolated," "purified," or "biologically pure" refer to the degree to which a material is free from components normally associated with it when found in its native state. "Isolate" refers to some degree of separation from its original source or surroundings. "Purity" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other materials so that any impurities do not substantially affect the biological properties of the protein or cause other adverse effects. That is, a nucleic acid or peptide of the invention is purified if it is substantially free from cellular material, viral material, or culture medium if produced by recombinant DNA techniques, or chemical precursors or other chemicals if chemically synthesized. Purity and homogeneity are generally determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can refer to the nucleic acid or protein giving rise to essentially one band in an electrophoretic gel. For proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications can give rise to different isolated proteins that can be purified separately.

[0038] An "isolated polynucleotide" refers to a nucleic acid (e.g., DNA) that is free of the genes that flank the gene in the naturally occurring genome of the organism from which the nucleic acid molecule of the present disclosure is derived. Thus, the term includes, for example, recombinant DNA that is incorporated into a vector; an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion). Furthermore, the term encompasses RNA molecules transcribed from DNA molecules, as well as recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0039] "Isolated polypeptide" refers to a polypeptide of the present disclosure that has been separated from components that naturally accompany it. Generally, a polypeptide is isolated if it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, preparations are at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, polypeptide of the present disclosure. Isolated polypeptides of the present disclosure can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0040] The term "hemogenic endothelial cells," as used herein, refers to cells that can differentiate to give rise to hematopoietic or endothelial cell types and can be derived from pluripotent stem cells as needed. Hemogenic endothelial cells are typically adhesive to extracellular matrix proteins and / or other hemogenic endothelial cells, and in some embodiments can be characterized by expression of the markers CD31 and CD34.

[0041] By "marker" is meant any protein or other epitope having altered expression levels or activity that is associated with a trait or condition.

[0042] The term "megakaryocyte" (MK), as used herein, refers to a large (e.g., diameter ≥ 10 μm), polyploid hematopoietic cell that tends to give rise to platelet precursors and / or platelets. One morphological characteristic of mature MKs is the development of large polymorphic nuclei. Mature MKs may cease proliferation, but continue to increase in DNA content through endomitosis, in parallel with an increase in cell size.

[0043] The term "megakaryocyte progenitor cell" (preMK), as used herein, refers to a mononuclear hematopoietic cell that is committed to the megakaryocyte lineage and is a precursor to mature megakaryocytes. Megakaryocyte progenitor cells are typically found in (but are not limited to) bone marrow and other hematopoietic locations, but can also be generated from pluripotent stem cells, such as by further differentiation of hemogenic endothelial cells that are themselves derived from pluripotent stem cells.

[0044] The term "microparticles" refers to very small (<1 micron) phospholipid vesicles shed from megakaryocytes or other cells. Microparticles may contain genetic material such as RNA and may express extracellular markers of their parent cells. Megakaryocyte- and platelet-derived microparticles may play a role in numerous pathways, including hemostasis and inflammation.

[0045] The term "platelet" (PLT) refers to cells 1-3 microns in diameter that lack a nucleus but contain RNA. Platelets may express CD41, CD42b, and CD61 on the cell surface. Internally, platelets contain alpha granules and dense granules, which contain factors such as P-selectin and serotonin, respectively. Platelets also have an open canalicular system, which refers to channels that are pathways for the transport of extracellular materials into the cell and the release of materials from the granules into the extracellular environment. They primarily function in regulating hemostasis by participating in blood clotting, but have also been shown to have a role in inflammation.

[0046] The term "preplatelet" refers to cells 3-10 microns in diameter that lack a nucleus but contain RNA. Preplatelets are otherwise morphologically and ultrastructurally similar to platelets and constitute an intermediate cell stage produced by megakaryocytes that disintegrate by cytoskeletal rearrangement to form individual platelets.

[0047] The term "proplatelets" refers to cytosolic extensions from or just released from megakaryocytes. Proplatelets disassemble by cytoskeletal rearrangements to form individual preplatelets and platelets.

[0048] The term "pluripotent stem cells" includes embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells, as well as other stem cells that have the ability to form cells derived from all three germ layers of the body, regardless of the method by which they are derived. Pluripotent stem cells are functionally defined as stem cells that can have one or more of the following characteristics: (a) the ability to induce teratomas when transplanted into immunodeficient (SCID) mice; (b) the ability to differentiate into cell types of all three germ layers (e.g., the ability to differentiate into ectodermal, mesodermal, and endodermal cell types); or (c) the ability to express one or more markers of embryonic stem cells (e.g., the ability to express Oct4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, SSEA-5 surface antigen, Nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.).

[0049] The term "induced pluripotent stem cells" (iPS cells or iPSCs) refers to a type of pluripotent stem cell generated by reprogramming somatic cells by expressing a combination of reprogramming factors. iPSCs can be generated using fetal somatic cells, postnatal somatic cells, neonatal somatic cells, juvenile somatic cells, or adult somatic cells. Factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4 (sometimes referred to as Oct3 / 4), Sox2, c-Myc, and Klf4. In other embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4, Sox2, Nanog, and Lin28. In certain embodiments, at least two, three, or four reprogramming factors are expressed in somatic cells to reprogram them.

[0050] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the probability of a disorder or condition occurring in a subject who does not have the disorder or condition, but is at risk of or susceptible to it.

[0051] By "reduce" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0052] "Reducing cell death" means reducing the tendency or probability that a cell will die. Cell death can be apoptotic, necrotic, or by any other means.

[0053] By "reduced level" is meant that the amount of analyte in a sample is less than the amount of analyte in a corresponding control sample.

[0054] "Reference" means a standard or control condition.

[0055] By "specifically binds" is meant that the compound or antibody recognizes and binds to a polypeptide of the present disclosure, but does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample, that naturally contains the polypeptide of the present disclosure.

[0056] The term "subject" or "patient" refers to an animal that is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a human or a non-human mammal, such as a mammal, including a non-human primate, murine, bovine, equine, canine, ovine, or feline.

[0057] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder or its associated symptoms. It will be understood that treatment of a disorder or condition need not completely eliminate, if not prevent, the disorder, condition, or its associated symptoms.

[0058] "Comprises," "comprising," "containing," "having," and the like have the meanings given them in U.S. patent law and can mean "includes," "including," and the like; "consisting essentially of" or "consists essentially of" likewise have the meanings given them in U.S. patent law, and the terms are open-ended, allowing for more than what is recited to be present so long as the basic or novel characteristics recited are not changed by the presence of more than recited, but prior art embodiments are excluded.

[0059] As used herein, the term "or" is understood to be inclusive unless otherwise stated or clear from context. As used herein, the terms "a," "an," and "the" are understood to be singular or plural unless otherwise stated or clear from context.

[0060] Unless otherwise specified or clear from the context, the term "about" as used herein is understood to be within the normal tolerance in the art, for example, within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value. Unless otherwise clear from the context, all numerical values ​​presented herein are modified by the term about.

[0061] The recitation of a list of chemical groups in any definition of a variable herein includes a definition of that variable as a single group or combination of the listed groups. The recitation of an embodiment for a variable or aspect herein includes a definition of that embodiment as a single embodiment or in combination with any other embodiment or portion thereof.

[0062] Any composition or method presented herein can be combined with any one or more of the other compositions and methods presented herein.

[0063] megakaryocyte In humans, megakaryocytes are CD34-positive cells that are primarily found in the bone marrow. +MKs are derived from hematopoietic stem cells, but are also found in the yolk sac, fetal liver, and spleen during early development. During MK differentiation, MK precursors undergo a period of endonuclear division, which allows MKs to become polyploid through numerous cycles of DNA replication without cell division, resulting in polymorphic nuclei with up to 128n copies of DNA. As MKs increase in size, their transcriptional and proteomic profiles also refine, the number of highly specialized granules, including α-granules and dense granules, and the development of highly invaginated membrane systems are hallmarks of MK maturation and development.

[0064] To produce platelets, MKs migrate to the vicinity of blood vessels in the bone marrow, through which they elongate long structures called proplatelets into the circulation. These proplatelets function as assembly lines for platelet production, sequentially releasing large numbers of anucleated platelets from their terminal ends.

[0065] Although platelets are primarily responsible for clot formation at sites of active bleeding, it is becoming increasingly clear that platelets also play important roles in wound healing, angiogenesis, and innate immunity. In the stem cell-based therapeutic landscape, platelets are ideal early participants because: 1) there is high clinical demand due to their short shelf life; 2) they are anuclear and can be safely irradiated to kill any contaminating nucleated cells, thereby reducing the risk of teratoma formation; 3) the vast majority (>90%) of platelet transfusions do not require HLA or blood type matching, thereby facilitating large-scale production of allogeneic human pluripotent stem cells (hPSCs) for universal therapeutic use; 4) they have a short and well-characterized lifespan, thereby simplifying planned clinical trials; 5) they are easily transplanted; and 6) aseptic manufacturing is highly beneficial, as current donor-based platelet transfusions are inherently prone to bacterial and viral contamination.

[0066] Current platelet demand exceeds supply by approximately 20%, and unmet demand is expected to more than double by 2022 due to a growing and aging population creating a greater need for platelet-based procedures, new medications to reduce platelet counts, and an increase in the existing use of platelet transfusions to improve healing times. In the United States, the blood market effectively operates as an oligopoly, with the American Red Cross and America's Blood Centers each controlling nearly half the market, and HemeXcel being the third-largest major blood provider.

[0067] There are many other potential markets for preMKs and MKs and their growth factors. For example, preMKs, MKs, and their lysates can be used for cell culture, tissue regeneration, wound healing, drug delivery, and in the cosmetics industry for skin rejuvenation. Because megakaryocytes produced by the methods described herein contain many growth factors, they can be a source of therapeutic compositions and / or used for many therapeutic purposes. The present disclosure addresses these needs by establishing a scalable, current Good Manufacturing Practice (cGMP)-compliant commercial platform for producing human iPSC-derived human megakaryocytes and their products.

[0068] In vitro, MKs can be reasonably derived from a variety of primary source stem cells, for example, pluripotent stem cells, hematopoietic stem cells, or other stem cell types.

[0069] In some embodiments, MKs can be derived from pluripotent stem cells, including, but not limited to, embryonic stem cells (ESCs) (e.g., human embryonic stem cells) and induced pluripotent stem cells (iPSCs) (e.g., human induced pluripotent stem cells). ESCs are pluripotent stem cells derived from the inner cell mass of an early preimplantation embryo called a blastocyst. iPSCs are a type of pluripotent stem cell that can be generated from adult cells by inducing the timed expression of specific transcription factors. iPSCs can be expanded indefinitely in culture, maintained, and manipulated to produce MKs.

[0070] In some embodiments, MK can be selected from, but not limited to, CD34 + Umbilical cord blood stem cells (UCB cells) (e.g., human CD34 + cord blood stem cells), CD34 + Mobilized peripheral blood cells (MPB cells) (e.g., CD34 + Hematopoietic stem cells, including human mobilized peripheral blood, or CD34+ bone marrow cells, can be derived from UCB cells, which are multipotent stem cells derived from the blood and remain in the placenta and attached umbilical cord after delivery. MPB cells are multipotent stem cells derived from volunteers whose stem cells have been mobilized into the bloodstream by administration of G-CSF or a similar agent.

[0071] In some embodiments, MKs can be derived from other stem cell types, including, but not limited to, mesenchymal stem cells (MSCs) (e.g., adipose-derived mesenchymal stem cells (AdMSCs)) or mesenchymal stem cells from other sources.

[0072] AdMSCs are derived from white adipose tissue, which is derived from the mesoderm during embryonic development and is present in all mammalian species and located throughout the body. Due to their wide availability and ability to differentiate into other tissue types of the mesoderm, including bone, cartilage, muscle, and fat, ASCs can be useful in a wide variety of applications.

[0073] In the present disclosure, stem cell cultures are maintained independently of embryonic fibroblast feeder cells and / or animal serum, which potentially contaminate with xenogeneic pathogens and increase the risk of immunogenic reactions in humans. Thus, the present method utilizes serum-free, feeder-free alternatives to avoid the introduction of animal products into the preMKs and MKs derived according to the present method, ensuring safe, animal-product-free conditions and products.

[0074] Production method Figure 1 shows an overall schematic diagram for the scalable differentiation of one or more pluripotent stem cells into megakaryocyte progenitor cells (preMKs), megakaryocytes (MKs), and platelets (PLTs). However, it should be noted that while the process is described in the context of pluripotent stem cells, in various embodiments, pluripotent stem cells can be replaced or supplemented with other types of stem cells.

[0075] Phase 0: Expansion and differentiation preparation of human induced pluripotent stem cells Matrix-dependent expansion culture For matrix-dependent expansion culture, clinical-grade pluripotent stem cells (PSCs) can be expanded as colonies by culturing them in pluripotent stem cell culture medium on a support matrix without feeder cells. The support matrix can be a two-dimensional surface or a three-dimensional structure. In some embodiments, the clinical-grade human induced pluripotent stem cells can be human induced pluripotent stem cells (iPSCs), such as PBG1, PBG2, or PBG3, although other types of pluripotent stem cells, such as embryonic stem cells, or other stem cells can also be used.

[0076] In some embodiments, the support matrix may be, by way of non-limiting example, recombinant vitronectin, recombinant laminin, Matrigel, or any combination of the foregoing. In some embodiments, the pluripotent stem cell culture medium may be, for example, but not limited to, Essential 8 Medium (ThermoFisher), StemFlex Medium (ThermoFisher), NutriStem Medium (Biological Industries), or other media capable of supporting the maintenance and growth of pluripotent cells known in the art. In some embodiments, cells may be cultured until confluency is reached. In some embodiments, cells may be cultured until 30% to 90% confluency is reached. In some embodiments, cells are cultured until up to 60%, up to 65%, up to 70%, or up to 75% confluency is reached. For example, cells are cultured until they reach approximately 70% confluency. Once a predetermined maximum percent confluency is reached, the cells are harvested. In some embodiments, cells can be collected as clumps by dissociation using 0.1 mM to 5 mM EDTA or a similar chelating agent or reagent. For example, cells can be collected using about 0.5 mM EDTA. In some embodiments, cells can be collected as single cells, for example, by dissociation using proteolytic enzymes, collagenolytic enzymes, or a combination thereof. For example, cells can be collected as single cells by dissociation using recombinant trypsin, such as TrypLE™ or Accutase™. For PSC maintenance / expansion, collected cells can be resuspended in pluripotent stem cell culture medium.

[0077] Matrix-independent 3D expansion culture For matrix-independent 3D expansion culture, clinical grade PSCs can be expanded as self-aggregating spheroids.In some embodiments, this can be achieved by seeding single cells at a density of about 100,000 to about 1.5 million per ml.For example, in some embodiments, single cells can be seeded at a density of 500,000 per ml.

[0078] Cells can be cultured in pluripotent stem cell culture medium under low- or non-adherent conditions, with continuous movement by slow stirring or gentle shaking. In some embodiments, a feeder-free, serum-free medium can be used. The pluripotent stem cell culture medium can be, for example, but not limited to, Essential 8 Medium (ThermoFisher), StemFlex Medium (ThermoFisher), NutriStem Medium (Biological Industries), or other similar media capable of supporting the maintenance and growth of pluripotent cells known in the art. In some embodiments, PSC spheroids are cultured for approximately 5-7 days until an overall cell density of about 3 to about 10 million cells per ml is reached and / or a median spheroid size of about 150 to about 350 μm is achieved. In some embodiments, PSC spheroids are cultured until an overall cell density of 5 million cells per ml is reached. In some embodiments, PSC spheroids are cultured until the cells reach a median spheroid size of approximately 250 μm. The culture step can last for 4, 5, 6, 7, or 8 days. If applicable, PSCs can be recovered as single cells by dissociation using proteolytic enzymes, collagenolytic enzymes, or a combination thereof. For example, cells can be recovered as single cells by dissociation using, but not limited to, trypsin, recombinant trypsin, such as TrypLE™, Accutase™, or similar reagents known in the art. In some embodiments, single cells are used to initiate another 3D expansion culture and / or directed differentiation culture.

[0079] Differentiation preparation In some embodiments, to prepare for differentiation, PSC aggregates can be generated by partially dissociating PSC colonies from matrix-dependent 2D cultures, by partially dissociating PSC spheroids from matrix-independent 3D cultures, or by self-aggregation of single PSCs generated by any method known in the art. In some embodiments, before initiating differentiation, these aggregates can be resuspended and cultured in pluripotent stem cell culture medium, such as, but not limited to, Essential 8 Medium (ThermoFisher), StemFlex Medium (ThermoFisher), or NutriStem Medium (Biological Industries). In some embodiments, the medium can contain a ROCK inhibitor, such as, but not limited to, Y27632, H1152, or a combination thereof. In some embodiments, the cells can be cultured at 37°C, 5% CO2, and 20% O2 for 0 to 72 hours before initiating differentiation.

[0080] For matrix-dependent culture, the aggregates can be allowed to attach to a surface. In some embodiments, the attachment step can proceed for about 24 hours, although any time between 1 hour and 24 hours or longer can be used. In some embodiments, the surface can be pre-coated with collagen, laminin, or any other extracellular matrix protein. In some embodiments, human type IV collagen can be used to coat the surface. In some embodiments, the matrix-coated surface can be 2D (e.g., the bottom of a plastic dish or flask). In some embodiments, the matrix-coated surface can be 3D (e.g., smooth or textured spherical microcarriers, macrocarriers, e.g., Raschig rings, etc.). Cells on the 3D matrix-coated surface can then be cultured with or without continuous movement. For example, cells can be cultured under ultra-low attachment static conditions in roller bottles, spinner flasks, stirred tank bioreactors, vertical wheel bioreactors, packed-bed bioreactors, or fluidized-bed systems.

[0081] For matrix-independent culture, the aggregates can be subjected to continuous movement by slow stirring or gentle shaking in a low-adhesion container. Between 0 and 72 hours, for example, after about 24 hours, the cells can be transitioned into phase I of differentiation.

[0082] Phase I. Generation of hemogenic endothelial cells In Phase I, the prepared PSCs can be differentiated into hemogenic endothelial cells. Briefly, the pluripotent stem cell culture medium is removed and replaced with Phase I differentiation medium. In some embodiments, the Phase I differentiation medium may be an animal component-free medium (ACF) containing StemSpan™-ACF (STEMCELL Technologies, Cat. No. 09855) as a basal medium, supplemented with one or more growth factors, including, for example, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF). In some embodiments, the basal medium is supplemented with one or more of BMP4 (e.g., 50 ng / ml), bFGF (e.g., 50 ng / ml), and VEGF (e.g., 50 ng / ml), each at between 1 ng / ml and 200 ng / ml. The cells can be incubated under hypoxic conditions (e.g., 37°C, 5% CO2, 5% O2) for 2 to 6 days, followed by incubation under normoxia (37°C, 5% CO2, 20% O2) for 2 to 6 days. In some embodiments, a WNT modulator, such as a WNT agonist or antagonist, can be added over the initial period of differentiation. In some embodiments, a GSK3 inhibitor can be added over the initial period of differentiation. In some embodiments, a GSK3 inhibitor or WNT agonist, such as CHIR9998014, CHIR99021, or a combination thereof, can be added over the initial period of differentiation, for example, for 1 to 2 days. In some embodiments, a WNT modulator can replace one or more of the growth factors over at least a portion of Phase I. For example, in some embodiments, while a WNT modulator is present, BMP4 can be added for the first 48 hours and may be unnecessary for the remainder of Phase I. In some embodiments, VEGF and bFGF may be unnecessary for the first 48 hours while a WNT modulator is present. In some embodiments, daily total medium changes can be performed throughout Phase I by removing spent medium and replacing it with fresh Phase I medium.In some embodiments, a partial medium exchange can be performed by removing 10-95% of the spent medium and replacing it with an equal volume of fresh Phase I medium. In some embodiments, an additional volume of fresh medium can be added, with the net effect of increasing the total volume of the culture. In some embodiments, instead of replacing or adding fresh Phase I medium, certain medium components are spiked into the culture.

[0083] In 2D matrix-dependent cultures, colony morphology changes to scattered, elongated cell clusters by day 2 (Figure 15). By days 5-6, a confluent, adherent layer of hemogenic endothelial cells is observed, with some three-dimensional structures within the adherent cell layer (Figure 15). In matrix-independent 3D cultures, spheroids grow larger, denser, and more heterogeneous as phase I progresses (Figure 33A). Approximately 6 days after the initiation of phase I differentiation, differentiation into hemogenic endothelium is complete. In some embodiments, differentiation can be considered complete when a confluent, adherent layer of hemogenic endothelial cells is observed, with some three-dimensional structures within the adherent cell layer (Figure 4). To confirm successful Phase I differentiation, a portion of the cells can be harvested as single cells using proteolytic enzymes, collagenolytic enzymes, or combinations thereof, such as Accutase® (STEMCELL Technologies, Cat. No. 07920), TrypLE Select® (Thermo Fisher Scientific, Cat. No. 12563029), or similar reagents known in the art, followed by flow cytometry analysis for the hemogenic endothelial-specific markers CD31 and CD34. In some embodiments, the hemogenic endothelial cells can also express CD309 and CD144 or CD309, CD144, CD140a, and CD235a. In some embodiments, Phase I can be performed in a stirred-tank bioreactor to form self-aggregating spheroids.

[0084] Phase II. Generation of exfoliated megakaryocyte progenitor cells (preMK) from hemogenic endothelial cells In some embodiments, megakaryocyte progenitor (phase II) differentiation can be initiated after Phase I for between 4 and 8 days. Briefly, Phase I medium is removed and replaced with an equal volume of Phase II medium, such as STEMdiff™ APEL™ 2 Basal Medium (STEMCELL Technologies, Cat. No. 05275). Such Phase II medium can be supplemented with one or more of the following: stem cell factor (SCF) (e.g., 25 ng / ml), thrombopoietin (TPO) (e.g., 25 ng / ml), Fms-related tyrosine kinase 3 ligand (Flt3-L) (e.g., 25 ng / ml), interleukin-3 (IL-3) (e.g., 10 ng / ml), interleukin-6 (IL-6) (e.g., 10 ng / ml), and heparin (e.g., 5 units / ml), at 1 ng / ml and 200 ng / ml, respectively. In some embodiments, the Phase II medium can be further supplemented with UM171, UM729, SR-1, SU6656, or any combination thereof.

[0085] The cells are then incubated at 37°C, 5% CO2, 20% O2 for at least 7 days and up to 12 days or more. For example, the cells can be incubated for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days. In some embodiments, daily partial medium changes can be performed by removing 10-95% of the spent medium and replacing it with an equal volume of fresh Phase II medium. In some embodiments, additional volumes of fresh medium can be added, with the net effect of increasing the total volume of the culture. In some embodiments, certain medium components can be spiked into the culture instead of replacing or adding fresh Phase I medium.

[0086] Within 2–3 days after the initiation of phase II, small, round, refractile cells appear within the adherent hemogenic endothelial cells and are eventually released into the supernatant (Figure 17A). These released cells may contain preMKs, defined by cell surface expression of CD43 and CD41 and the lack of CD14 expression. In 2D cultures, floating, weakly adherent phase II cells that appear on top of the adherent cell layer can be collected daily by gently rinsing and collecting the medium into a conical tube. Half-medium changes can be initiated by adding half the original volume of fresh phase II medium to the top of the rinsed adherent cell layer. An aliquot of cells collected in the medium can be removed for viable cell enumeration and biomarker analysis by flow cytometry. The remainder of the cells can then be centrifuged at 200 x g for 5 minutes. After centrifugation, medium changes can be completed by adding half the original volume of supernatant back to the adherent cell layer. In matrix-independent 3D culture, the released cells can be harvested by ceasing agitation, allowing the spheroids to settle to the bottom of the container, and then collecting up to 90% of the medium, along with the suspended cells, in a centrifuge tube (e.g., a conical tube). Half the original volume of fresh medium can then be added to the container, and conditioned medium from the supernatant of the centrifuged cells can be added. In some embodiments, an additional volume of fresh medium can be added, with the net effect of increasing the total volume of the culture. In some embodiments, instead of replacing or adding fresh Phase II medium, specific medium components can be spiked into the culture. The remainder of the supernatant can be discarded, and the cell pellet containing preMK can be stored at -180°C in Cryostor 10 cryopreservation medium or can be directly progressed to Phase III. Megakaryocyte progenitor cells collected during Phase II are small, round, refractile cells that express CD43 and CD41 but lack CD14 expression.

[0087] Phase III. Generation of mature megakaryocytes (MK) from megakaryocyte progenitor cells In some embodiments, differentiation of mature megakaryocytes can be initiated using PSC-derived preMKs generated as described above. Fresh or thawed megakaryocyte progenitor cells can be seeded on a non-adherent surface medium in Phase III, for example, containing StemSpan™-ACF. A non-adherent surface refers to a surface on which the majority of cells are not intended to stick or adhere, but instead remain largely in suspension. For example, such a surface may be made of "ultra-low adhesion plastic" or may not be coated with extracellular matrix proteins to prevent or minimize cell adhesion to the surface. In some embodiments, Phase III medium can be supplemented with one or more of TPO (e.g., 25 ng / ml), SCF (e.g., 25 ng / ml), IL-6 (e.g., 10 ng / ml), IL-9 (e.g., 10 ng / ml), and heparin (e.g., 5 units / ml), each at between 0 ng / ml and 200 ng / ml. In some embodiments, the Phase III medium can also be supplemented with UM171, UM729, SR-1, SU6656, or any combination thereof.

[0088] The cells can then be incubated for up to 5 days at between 37°C and 40°C (e.g., 39°C), between 5% and 20% CO2 (e.g., 7%-10%), and between 5% and 20% O2. In some embodiments, daily partial medium changes are performed by removing 10-95% of the spent medium and replacing it with an equal volume of fresh Phase III medium. In some embodiments, the non-adherent surface is a 6-well ultra-low attachment plate. In some embodiments, the non-adherent surface is a gas-permeable membrane (e.g., G-Rex®, etc.). In some embodiments, the non-adherent surface is a cell culture bag or container with gentle agitation.

[0089] In some embodiments, during phase III, megakaryocyte progenitor cells can be differentiated into mature MKs within a few days. In some embodiments, initially uniformly small, round, and refractile cells (Figure 21) begin to increase in size and ploidy by days 2-4 (Figure 20). Concurrently, MKs producing platelet precursors can be readily observed (Figure 20). By days 3-4 of phase III, CD61 co-expresses the mature MK markers CD42a and CD42b. + The proportion of (megakaryocyte-lineage) cells increases dramatically and can reach levels as high as 80-90%, depending on the starting hiSPC cell line (Figure 21). By days 4-5 of phase III, mature MKs release platelets into the culture medium. These platelets can be collected, quantified, and evaluated by flow cytometry and electron microscopy, thereby confirming their identity as bona fide platelets (Figure 45).

[0090] 3D system Packed Bed Bioreactor In some embodiments, 3D scalable packed-bed bioreactors can be used for the production of one or more of preMKs, megakaryocytes, platelets, or megakaryocytes and platelets. In some embodiments, packed-bed bioreactors can be used for Phase I and Phase II culture. For example, packed-bed bioreactors can be used to differentiate PSCs into hemogenic endothelial cells and then produce preMKs. Packed-bed reactor carriers can be micro- or macro-sized and made from biocompatible plastics, metals, glass, or natural materials such as alginate. In some embodiments, the carriers are made from PTFE and are shaped like Raschig rings, e.g., 1 mm Raschig rings. In some embodiments, the carriers can be coated with the above-mentioned matrices. In some embodiments, the carriers can be coated with laminin, e.g., recombinant human protein laminin 521. In some embodiments, pluripotent cells can be seeded onto the carriers as aggregates. In some embodiments, the medium can be removed and replaced with Phase I medium during daily medium changes. In some embodiments, during Phase I, pluripotent cells may exhibit growth regions inside the carriers in the packed-bed reactor. In some embodiments, initial differentiation of pluripotent cells into hemogenic endothelium (i.e., Phase I of directed differentiation), as well as further differentiation and release of preMK (i.e., Phase II of directed differentiation) can occur within the same vessel. For example, a packed-bed bioreactor can be used to culture pluripotent cells, e.g., PBG-1 The packed bed may contain laminin-521 coated macrocarriers seeded with iPSCs. The packed bed may then be exposed to a continuous flow of medium to allow phase I differentiation into hemogenic endothelium. After permeating the packed bed, the medium may be recirculated to the cells after circulating through a conditioning chamber where fresh medium components can be added and oxygen / CO2 concentrations can be adjusted by sparging or other means.

[0091] Upon completion of Phase I, the medium can be switched to allow for Phase II differentiation and the production and release of preMKs. Appropriately sized and shaped carriers, such as 1 mm Raschig rings, can allow for sufficient medium flow and channel width to allow released cells to permeate through the packed bed and out of the reactor for collection and cryopreservation. In some embodiments, this design can reduce shear forces experienced by cells, may allow for efficient medium use due to the perfusion-based design, and allows for the continuous collection of preMKs as they are released.

[0092] Self-aggregating spheroids in stirred tank bioreactors In some embodiments, scalable 3D solutions can be used to perform certain process steps that may involve performing differentiation using self-aggregating spheroids suspended in agitated or shaken vessels (Figure 32). In some embodiments, such vessels may include low- or non-adherent surfaces, i.e., surfaces coated with hydrophilic or neutrally charged coatings to inhibit specific and non-specific cell immobilization to the surface, thereby forcing the cells into suspension. Pluripotent cells can be dissociated into single cells and resuspended in pluripotency maintenance medium. In some embodiments, the maintenance medium can be supplemented with a Rock inhibitor, such as H1152 or other Rock inhibitors. The pluripotent cells can then be incubated in the low- or non-adherent vessels and subjected to agitation under standard culture conditions (e.g., 37°C, 5% CO2, 20% O2). In some embodiments, the incubation vessel can be placed on an orbital shaker to provide agitation, or shaker or spinner flasks with constant agitation, or controlled stirred-tank bioreactors can be used. Within 24 hours, the pluripotent cells can self-aggregate to form spheroids with a diameter of approximately 50-150 μm. When stirring is stopped, the spheroids can settle to the bottom of the container.

[0093] The medium can then be replaced with Phase I differentiation medium to promote differentiation into hemogenic endothelium, and agitation can be resumed along with incubation under hypoxic conditions (e.g., 37°C, 5% CO2, 5% O2). Medium changes can be performed periodically (e.g., daily) during which time the spheroids can grow larger and develop characteristic structures and shapes. For example, as shown in Figure 33A, spheroids can be cultured for a total of 6 days (4 days at 37°C, 5% CO2, 5% O2, followed by 2 days at 37°C, 5% CO2, 20% O2). As shown in Figure 33A, on day 6, the spheroids are larger, denser, and have an irregular surface.

[0094] To transition to Phase II, agitation can be stopped and the spheroids can be allowed to settle to the bottom of the container. The medium can then be replaced with Phase II differentiation medium to promote differentiation and release of the suspended cells. Thereafter, the suspended cells can be collected periodically (e.g., daily), and a partial medium exchange can be performed. The medium can be collected and centrifuged. Approximately half the working volume of fresh Phase II differentiation medium can be added to the spheroids along with a sufficient volume of conditioned medium (i.e., the supernatant after centrifugation) to return them to the original working volume. The cell pellet can be cryopreserved or can be transitioned to Phase III for maturation into mature MKs.

[0095] Upon transition to static phase III culture, preMKs from 3D self-aggregating spheroid cultures can yield MK purity similar to that of preMKs from 2D culture systems. Furthermore, phase III differentiation cultures generated from 3D self-aggregating spheroid cultures dramatically increase in size and can contain cells capable of generating platelet precursors, consistent with their identity as bona fide megakaryocytes.

[0096] Moving to a scalable system for Phase III In some embodiments, as described above, fresh or thawed megakaryocyte preMKs can be seeded onto a low- or non-adherent surface in Phase III medium. In some embodiments, such a non-adherent surface can be a gas-permeable membrane (such as G-Rex®). In some embodiments, the low- or non-adherent surface is a cell culture bag or container with gentle agitation. In either case, preMKs (either freshly harvested from Phase II cultures or thawed from frozen stocks) are suspended in Phase III medium at a density of 0.5 to 10 million cells per ml and introduced into the container. For example, preMKs may be at a density of 1-1.5 million cells per ml, 1-2 million cells per ml, 1-3 million cells per ml, 1-4 million cells per ml, 2-5 million cells per ml, 2-6 million cells per ml, 3-7 million cells per ml, 3-8 million cells per ml, 5-9 million cells per ml, or 8-10 million cells per ml. Cells are cultured for a total of 1-5 days (e.g., 3 days) to allow differentiation into mature MKs. In some embodiments, daily half-medium changes are performed by removing 10-95% of the spent medium and replacing it with an equal volume of fresh Phase III medium. At the end of Phase III culture, the resulting cells have increased in size and ploidy and exhibit many features indicative of mature megakaryocytes (shown, by way of example, in Figures 34-42 and described below).

[0097] Megakaryocytes and their products In some embodiments, the present disclosure provides megakaryocyte progenitor cells, megakaryocytes, pre-platelets, platelet precursors, or platelets derived in vitro from PSC cells or cell lines. According to aspects of the present disclosure, megakaryocyte progenitor cells, megakaryocytes, pre-platelets, platelet precursors, or platelets derived from PSC cells or cell lines are produced using the methods of U.S. Patent No. 9,763,984 or the bioreactor disclosed in International Application No. PCT / US2018 / 021354, which are incorporated by reference in their entireties.

[0098] In some embodiments, the present disclosure provides an isolated population of cells comprising megakaryocytes or megakaryocyte progenitor cells.

[0099] In some embodiments, the present disclosure provides compositions containing megakaryocytes or megakaryocyte progenitor cells. In some embodiments of the present disclosure, compositions comprising megakaryocytes, megakaryocyte progenitor cells, or products thereof are disclosed.

[0100] According to some embodiments of the present disclosure, megakaryocytes, megakaryocyte progenitor cells, or their products are homogeneous in shape, size, and / or phenotype.It should be understood that the megakaryocytes, megakaryocyte progenitor cells, or their products of the present disclosure may contain variability in biomarker expression, size, ploidy, number, and purity that is characteristically different from the variability in corresponding human cells.In some embodiments, such variability can be significantly reduced.In some embodiments, cell populations can be created to have a desired variability that is lower or higher than the variability of naturally occurring cells.

[0101] In some embodiments, megakaryocyte progenitor cells (preMK) are characterized by the expression of the markers CD43 and CD41, and the absence of CD14 (i.e., CD14 - , CD41 + , CD43 + ) The additional expression of CD42b may indicate that the megakaryocyte progenitor cells are in the process of final maturation into mature megakaryocytes. In certain embodiments, megakaryocyte progenitor cells generated under differentiation culture are non-adherent and can float freely in the culture medium.

[0102] In some embodiments, the megakaryocytes are CD42a + , CD42b + , CD41 + , CD61 + , GPVI + , and DNA + In some embodiments, the megakaryocytes are one or more of CD42a + , CD42b + , CD41+ , CD61 + , and DNA + In some embodiments, the megakaryocytes are one or more of CD42b + , CD61 + , and DNA + In some embodiments, the megakaryocytes are one or more of CD42a + , CD61 + , and DNA + In some embodiments, the megakaryocytes are one or more of CD42a + , CD41 + , and DNA + In some embodiments, the megakaryocytes are one or more of CD42b + , CD41 + , CD61 + , and DNA + In some embodiments, the megakaryocytes are one or more of CD42b + , CD42a + , CD61 + , and DNA + In some embodiments, the megakaryocytes are one or more of: CD42b + , CD42a + , CD41 + , and DNA + In some embodiments, the megakaryocytes are one or more of: CD41 + CD61 + CD42b + GPVI + In some embodiments, the megakaryocytes are CD41 + CD61 + CD42a + GPVI + is.

[0103] In some embodiments, the megakaryocytes are CD61 + and DNA +and have a diameter of about 10-50 μm. In some embodiments, the average size of megakaryocytes produced by the methods described herein is between 10 μm and 20 μm, between 11 μm and 19 μm, between 12 μm and 18 μm, between 13 μm and 17 μm, between 14 μm and 16 μm, or between 14 μm and 15 μm. In some embodiments, the average size of megakaryocytes produced by the methods described herein is 14.5 μm. In some embodiments, the diameter of the megakaryocytes is about 10-20 μm. In some embodiments, the diameter of the megakaryocytes is about 10-30 μm. In some embodiments, the diameter of the megakaryocytes is about 10-40 μm. In some embodiments, the diameter of the megakaryocytes is about 10-50 μm. In some embodiments, the diameter of the megakaryocytes is about 20-40 μm. In some embodiments, the megakaryocytes have a diameter of about 25-40 μm.

[0104] In some embodiments, the megakaryocytes produced by the methods described herein have a ploidy of 2N to 16N. In some embodiments, the megakaryocytes have a ploidy of at least 4N, 8N, or 16N. In some embodiments, the megakaryocytes have a ploidy of 4N to 16N. In some embodiments, the megakaryocytes produced by the methods described herein have a ploidy of 16% + / - 11.4% CD61+ cells at 72 hours of Phase III culture, which is higher than 4N DNA.

[0105] In some embodiments, at least 50% of the megakaryocyte population produced by the methods described herein is CD61 + and DNA + and ploidy ranges from 2N to 16N. For example, megakaryocytes (i.e., beta-1-tubulin-positive phase III cells) from a representative PBG1 differentiation culture range in size from approximately 9 μm to approximately 27 μm, with a median size of 15 μm. This average size is similarly comparable to "normal" megakaryocytes derived from various bone marrow sources (Figure 41).

[0106] In some embodiments, the isolated cell population or composition is at least 50% CD42b + CD61 +DNA + In some embodiments, the isolated cell population or composition contains at least 55% CD42b + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 65% CD42b + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 60% CD42b + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 70% CD42b + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 75% CD42b + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 80% CD42b + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 85% CD42b + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 90% CD42b + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 95% CD42b + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 98% CD42b + CD61 + DNA + Contains cells.

[0107] In some embodiments, the isolated cell population or composition is at least 50% CD42b + CD41 + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 55% CD42b + CD41 + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 65% CD42b + CD41 + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 60% CD42b + CD41 + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 70% CD42b + CD41 + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 75% CD42b + CD41 + CD61 + In some embodiments, the isolated cell population or composition contains at least 80% CD42b + CD41 + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 85% CD42b + CD41 + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 90% CD42b + CD41 + CD61 +DNA + In some embodiments, the isolated cell population or composition contains at least 95% CD42b + CD41 + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 98% CD42b + CD41 + CD61 + DNA + Contains cells.

[0108] In some embodiments, the isolated cell population or composition is at least 50% CD42b + CD42a + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 55% CD42b + CD42a + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 65% CD42b + CD42a + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 60% CD42b + CD42a + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 70% CD42b + CD42a + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 75% CD42b + CD42a + CD61 + DNA +In some embodiments, the isolated cell population or composition contains at least 80% CD42b + CD42a + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 85% CD42b + CD42a + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 90% CD42b + CD42a + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 95% CD42b + CD42a + CD61 + DNA + In some embodiments, the isolated cell population or composition contains at least 98% CD42b + CD41 + CD61 + DNA + Contains cells.

[0109] In some embodiments, the isolated cell population or composition contains at least 50% megakaryocytes with a ploidy of 4N or greater. In some embodiments, at least 50% of the megakaryocytes have a ploidy of 4N to 16N. In some embodiments, at least 60% of the megakaryocytes have a ploidy of 4N to 16N. In some embodiments, at least 70% of the megakaryocytes have a ploidy of 4N to 16N. In some embodiments, at least 80% of the megakaryocytes have a ploidy of 4N to 16N. In some embodiments, at least 90% of the megakaryocytes have a ploidy of 4N to 16N. In some embodiments, the isolated cell population or composition contains megakaryocytes with an average ploidy of 4N.

[0110] In some embodiments, the isolated cell population or composition contains platelet precursors, preplatelets, or platelets generated from megakaryocytes of the present disclosure. In some embodiments, the platelet precursors, preplatelets, or platelets express CD42b + CD61 + DNA - In some embodiments, megakaryocytes are produced by differentiation of hiPSC cells or cell lines in vitro.

[0111] In some embodiments, megakaryocytes produced by the methods described herein comprise one or more of the following: (a) MK granule content by immunofluorescence microscopy: PF4 and VFW for alpha granules, LAMP-1 and serotonin for dense granules; (b) gene expression data: Oct4-, Nanog-, Sox2-, Zfp42-, Zfpm1+, Nfe2+, Runx1+, Meis1+, Gata1+; (c) low fibrinogen, serotonin, and LDL / absence of fibrinogen, serotonin, and LDL, and (d) capable of taking up fibrinogen, serotonin, and LDL when incubated with plasma.

[0112] In some embodiments, megakaryocytes produced by the methods described herein have a characteristic expression profile of growth factors, cytokines, chemokines, and related factors (Figure 44). In some embodiments, the present disclosure provides compositions or pharmaceutical compositions comprising the megakaryocytes, which may contain factors such as platelet-derived growth factor isoforms PDGF-AA or PDGF-BB, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), hematopoietic growth factors Flt3L, G-CSF, GM-CSF, interleukins (IL-1RA, IL-8, or IL-16), C-X-C chemokine family members CXCL1 (GROalpha) or CXCL12 (SDF-1), TNF superfamily members sCD40L or TRAIL, or C-C chemokine family members CCL5 (RANTES), CCL11 (eotaxin-1), CCL21 (6CKine), or CCL24 (eotaxin-2). In some embodiments, the present disclosure provides a composition or pharmaceutical composition comprising the lysate of the megakaryocyte. Such lysate can be prepared by any method known in the art, for example, by disrupting the membrane of preMK or MK by viral, enzymatic, or osmotic mechanisms that damage its integrity. In some embodiments, the lysate can contain additional agents or be prepared as different compositions (liquid, paste, etc.) according to the needs of specific applications. In some embodiments, such compositions may include factors such as platelet-derived growth factor isoforms PDGF-AA or PDGF-BB, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), hematopoietic growth factors Flt3L, G-CSF, GM-CSF, interleukins (IL-1RA, IL-8, or IL-16), C-X-C chemokine family members CXCL1 (GROalpha) or CXCL12 (SDF-1), TNF superfamily members sCD40L or TRAIL, or CC chemokine family members CCL5 (RANTES), CCL11 (eotaxin-1), CCL21 (6CKine), or CCL24 (eotaxin-2).

[0113] How to use In some embodiments, the present preMK and MK and their components can be a source of growth factors, such as human growth factors. In some embodiments, such growth factors can be used for cell culture, tissue regeneration, wound healing, bone regeneration, functional cosmetics, and hemostatic bandages. In some embodiments, the present megakaryocytes, their lysates, or compositions thereof can be used for cell culture. In some embodiments, the present megakaryocytes, their lysates, or compositions thereof can be used as functional cosmetics. In some embodiments, the present megakaryocytes, their lysates, or compositions thereof can be used as therapeutic agents. For example, the present megakaryocytes, their lysates, or compositions thereof can be used to increase cell expansion ex vivo, improve bone marrow regeneration in vivo, increase tissue regeneration and angiogenesis, and increase animal survival rates in radiation studies.

[0114] In some embodiments, the present pre-MKs and MKs can be used to generate platelets to support current transfusion needs (e.g., surgery, chemotherapy, pregnancy / childbirth, trauma). National defense and security initiatives are a high priority within the United States and represent a large potential market for MKs and resulting products as radiation countermeasures. Radiation exposure, such as that occurring after a nuclear accident or attack, inhibits platelet production. A major radiological event would trigger an immediate demand for platelets that would deplete existing local stockpiles for treating emergency trauma, followed by a sustained demand for platelets in survivors 6+ days after exposure. As our readiness gap shifts from the front lines to 24-48 hours after the event, when affected populations become thrombocytopenic, a national strategic stockpile of platelets becomes critically important. The United States does not maintain a platelet inventory in the Strategic National Stockpile, and no therapeutics are approved to rapidly increase platelet counts. According to some embodiments, pre-MKs and MKs can be used for on-demand platelet production. The ability to bank pre-MKs over the long term and develop on-demand hiPSC-platelet production capacity will enable the establishment of a strategic national stockpile of hiPSC-platelets, which will be crucial to meeting this anticipated need.

[0115] Autologous platelet-rich plasma (PRP)-supplemented media has been shown to nourish microvascular endothelial cells and improve the preservation of vascular integrity in organs perfused for transplantation. Platelets store bioactive factors acquired from megakaryocytes in secretory granules. These include various chemokines and growth factors, such as platelet-derived growth factor isoforms (PDGF-AA, -AB, and -BB), transforming growth factor-b (TGF-b), insulin-like growth factor 1 (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF or FGF-2), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), and bone morphogenetic protein 2, bone morphogenetic protein 4, and bone morphogenetic protein 6 (BMP-2, BMP-4, BMP-6). Human platelet lysate dramatically increases ex vivo cell expansion, improves in vivo bone marrow regeneration, and increases animal survival in radiation studies. In some embodiments, the present disclosure provides compositions or pharmaceutical compositions comprising a lysate of platelet precursors, preplatelets, or platelets generated from the megakaryocytes, which may include factors such as the platelet-derived growth factor isoforms PDGF-AA or PDGF-BB, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), the hematopoietic growth factors Flt3L, G-CSF, GM-CSF, interleukins (IL-1RA, IL-8, or IL-16), the C-X-C chemokine family members CXCL1 (GROalpha) or CXCL12 (SDF-1), the TNF superfamily members sCD40L or TRAIL, or the C-C chemokine family members CCL5 (RANTES), CCL11 (eotaxin-1), CCL21 (6CKine), or CCL24 (eotaxin-2).

[0116] kit The present disclosure provides a kit comprising the megakaryocytes or differentiated cells of the present disclosure. In one embodiment, the kit comprises a composition comprising isolated megakaryocytes. In certain embodiments, the present disclosure provides a kit for differentiating, culturing, and / or isolating the megakaryocytes or their precursors of the present disclosure. In certain embodiments, the present disclosure provides a kit for producing platelets.

[0117] In some embodiments, the kits include a sterile container for containing the cell composition, which may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceuticals.

[0118] If desired, the kit is provided with instructions for generating megakaryocytes. The instructions generally include information regarding the conditions and factors necessary for differentiation, culture, and / or isolation of megakaryocytes or their precursors. In some embodiments, instructions for producing platelets are included. The instructions may be printed directly on the container (if present), printed as a label affixed to the container, or printed as a separate sheet, booklet, card, or folder provided in or with the container.

[0119] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the capabilities of those skilled in the art. Such techniques are incorporated herein by reference in their entirety. Laboratory Manual”, second edition (Sambrook, 1989);”Oligonucleotide Synthesis” (Gait, 1984);”Animal Cell Culture” (Freshney, 1987);”Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996);”Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987);”Current Protocols in Molecular Biology” (Ausubel, 1987);”PCR: The Polymerase Chain These techniques are applicable to the production of the polynucleotides and polypeptides of the present disclosure and therefore may be considered in making and practicing the present disclosure. Techniques particularly useful for certain embodiments are discussed in the following sections.

[0120] The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and treatment methods of the present disclosure, and are not intended to limit the scope of what the inventors consider to be their disclosure. [Example]

[0121] Example 1 Expansion of megakaryocytic progenitor cells, megakaryocytes, and platelets from clinical-grade hiPSC cell lines Clinical-grade hiPSC cell lines were tested for their potential to differentiate into megakaryocytes using the directed differentiation protocol shown in Figure 2. Figure 2 is a schematic diagram showing the time course of differentiation from pluripotent stem cells to megakaryocytes, with each phase of differentiation (phases 0, I, II, and III) indicated. Cell types and cell markers in phases 0–III are indicated above the timeline. Culture conditions, including media composition, matrix, temperature, and gas conditions, are indicated below the timeline.

[0122] Different iPSC lines are functionally distinct, and to truly optimize differentiation protocols, it is important to identify clinical-grade cell lines that are optimal for the process. Three clinical-grade hiPSC cell lines, designated PBG1, PBG2, and PBG3, were obtained. PBG1 was obtained from the NINDS Human Cell Imaging Center at the National Institute of Neurological Disorders and Stroke (NINDS) / National Institutes of Health (NIH). PBG1 (NINDS ID: LiPSC-Gr1.1) was obtained from the National Center for Clinical Data Repository (NHCDR). + PBG2 and PBG3 were derived from umbilical cord blood (Lonza). PBG2 and PBG3 were obtained from Fujifilm-Cellular Dynamics International (F-CDI). PBG2 and PBG3 were derived from male and female adult blood cells, respectively (F-CDI MyCells iPSC ID numbers: 21525 and 21526).

[0123] Prior to the initiation of differentiation, all three hiPSC lines formed characteristic colonies when grown on vitronectin using Essential 8 Medium (Figure 3A) and displayed expression of the pluripotency biomarkers OCT4 and NANOG (Figure 3B, Figure 13A). Using the multiphase protocol summarized in Figure 2, three clinical-grade hiPSC cell lines were directed to differentiate into megakaryocytes. Cells were observed at various time points during directed differentiation (Figures 4A-4D). CD31, a marker of hemogenic endothelium, was also expressed. + The differentiation efficiency during Phase I was first observed by monitoring the expression of CD41 (Figure 5). Differentiation efficiency was similar for the three hiPSC cell lines during Phase I. Without being bound by theory, the differences observed in megakaryocyte output / quality at later stages during the differentiation process did not necessarily correlate with differentiation efficiency in Phase I. In Phase II, CD41 +CD43 + By monitoring cell production, further differentiation into megakaryocyte progenitors was observed (Figure 6A). Notably, for some clinical-grade cell lines, megakaryocyte progenitor production during Phase II was prolonged (up to 17 days) compared to previous methods using different hiPSC cell lines (Figure 6B). Cumulative yields were particularly robust for PBG-1 and PBG-2 iPSCs, showing some variability between independent differentiations (Figure 6C). Transitioning cells to Phase III of the directed differentiation protocol resulted in the upregulation of CD42b + Expression of CD42b increased from day 1 to day 5, and from day 4 to day 5, + expressing CD41 + The percentage of cells was at least approximately 80% (Figure 7A). Phase III cells derived from the three clinical-grade iPSC lines were analyzed by light, immunofluorescence, and electron microscopy, which revealed that PBG1 and PBG2 displayed characteristics consistent with those of mature megakaryocytes, including size, morphology, proplatelet elongation, megakaryocyte-specific protein expression, and ultrastructure (Figures 7B-7C, 8A-8E).

[0124] Example 2 Large-scale expansion of pluripotent PBG-1 cells To generate the large numbers of cells required for high-density seed banks and to generate sufficient cell numbers to initiate differentiation at a scale reasonable for clinical production, pluripotent PBG-1 cells must be expanded prior to differentiation. PBG-1 cells can be maintained and expanded in 2D culture using recombinant vitronectin (VTN) and additional animal component-free (ACF) cGMP-compliant reagents, such as Essential 8, NutriStem, or StemFlex. Characteristic colony growth and maintenance of pluripotency markers were observed in all three growth conditions (Figures 9A–9C, 10A–10C). To enable large-scale expansion, PBG-1 cells were harvested as single cells from 2D culture using TrypLE and allowed to self-aggregate in a stirred 3D vessel, in this case a 300 ml DasBOX mini bioreactor system. A ROCK inhibitor, such as Y27632, was added to the cells for the first 24 hours to promote cell survival during initial aggregation over 6–7 days in a stirred tank; the resulting spheroids increased in diameter from 50 to 250 microns and achieved up to a 40-fold increase in overall cell density during that time (Figures 11A–11C). PBG-1 cells grown in this manner could be passaged repeatedly, maintained pluripotency over at least four successive rounds of expansion (Figures 12A–12B, 13B), and maintained a normal karyotype (Figure 14).

[0125] Example 3 Detailed characterization of PBG-1 directed differentiation into preMKs and MKs using type IV collagen matrix in 2D culture vessels PBG-1 cells were harvested using 0.5 mM EDTA and collected as small clumps at 4.2 μg / cm 2When plated on human type IV collagen, they exhibit a characteristic series of morphological changes over the course of 6 days of phase I differentiation (Figure 15). At the end of phase I, representative wells are harvested as single cells using Accutase and assessed for the hemogenic endothelial markers CD31 and CD34 by flow cytometry (Figure 16A). Across multiple independent PBG-1 differentiations (n=41), the average differentiation efficiency at day 6 was determined to be approximately 40% CD31+ (range: approximately 20-60%) and approximately 30% CD31+CD34+ (range, approximately 15%-45%) (Figure 16B).

[0126] Within 2–3 days after the initiation of phase II (i.e., day 6+2–day 6+3), small, round, refractile cells appear within the adherent hemogenic endothelial cells and are eventually released into the supernatant above the adherent hemogenic endothelial monolayer (Figure 17A). These released cells contain preMKPs, defined by cell surface expression of CD43 and CD41, and the absence of CD14 expression (Figure 17B, Figure 17C). Floating, weakly adherent phase II cells appearing on top of these adherent cell layers are collected daily by gently rinsing and collecting the medium into conical tubes and analyzed daily for CD43, CD41, and CD14 expression. The purity of the released cells is low during the first few days of phase II and then plateaus, with an average peak preMKP purity of 50–60% by day 6+6 (Figure 18A). CD14+ myeloid cells were not a major contaminant in PBG-1 directed differentiation cultures during the first 6–7 days of phase II, but there was some variability thereafter (Figure 18B). The kinetics of preMK production peaked on average at days 6+6 and 6+7 and then declined (Figure 19A). Across multiple independent PBG-1 differentiations (n ​​= 41), the mean cumulative preMK (CD43+CD41+CD14-) yield was determined to be approximately 1 million cells per well (range: 0.1–3.3 million) (Figure 19B).

[0127] When preMKs from these cultures are transferred to phase III conditions, they differentiate into mature MKs within a few days. Initially, the cells are uniformly small, round, and refractile (Figure 20A) and begin to increase in size by days 2-4 (Figures 20B and 20C). Concurrently, MKs producing platelet precursors can be readily observed (Figures 20C and 20D). By days 3-4 of phase III, CD61 co-expresses the mature MK markers CD42a and CD42b. + The percentage of (megakaryocyte lineage) cells was determined by FACS (Figures 21A and 21B), and the purity of mature MK (CD61+CD42a+CD42b+ cells) could reach levels as high as 70-90% of all nucleated cells in the culture (Figure 21C).

[0128] Example 4 To support directed differentiation of PBG-1 to MK, type IV collagen can be replaced with recombinant laminin-521. Type IV collagen was purified from human placental material and used for research purposes. Type IV collagen is available only as a reagent (Cat. No. 10011001). Therefore, type IV collagen is not compatible with cGMP production of PBG-1-derived megakaryocytes, and alternative strategies must be developed before these cells can be used clinically. One potential solution is to replace type IV collagen with recombinant matrix components produced from recombinant sources. Here, we demonstrate that recombinant laminin-521 can be used as a suitable alternative to type IV collagen for directed differentiation of iPSCs to MKs. PBG-1 aggregates generated by harvesting with 0.5 mM EDTA exhibited a density of 0.13 μg / cm. 2 During the course of 6 days of Phase I differentiation with recombinant laminin-521 at 4.2ug / cm 2The cells exhibited the same characteristic series of morphological changes as those in human type IV collagen (Fig. 22A-22B). Upon transition to phase II, laminin-521 cultures produced preMKs of similar yield and purity as the corresponding type IV collagen cultures (Fig. 23A-23C). Upon additional differentiation for 3 days, cells in phase III, whether produced in type IV collagen or laminin-521, exhibited similar size, morphology, and propensity for the production of platelet precursors (Fig. 24A-24B). They co-expressed CD61, which is a mature MK marker, CD42a and CD42b. + The percentage of cells (megakaryocyte lineage) was also measured and found to be similar for cells generated in either matrix (FIGS. 25A-25B).

[0129] Example 5 WNT modulators can affect phase I and II differentiation efficiency. WNT signaling is crucial during development. The GSK3 kinase inhibitors CHIR98014 and CHIR99021 act as WNT agonists. When the phase I differentiation conditions described herein (using a laminin-521 matrix) were enhanced with 0.6 μM CHIR98014 or 6 μM CHIR99021 for only the first 48 hours of differentiation, a dramatic increase in phase I differentiation efficiency was observed on day 6, as determined by immunofluorescent staining for CD31 and CD34 (Figures 26A-26C). Control and CHIR98014 cultures were then transitioned to phase II, where preMK production and release were tracked by immunofluorescent staining for CD41 and CD43. Visual estimation of the number of CD41+ cells suggests that the higher phase I efficiency generated by WNT modulators during the first 48 hours may correspond to higher output during phase II (Figures 27A-27B). Therefore, short-term addition of WNT modulators can affect differentiation efficiency throughout subsequent differentiation phases.

[0130] Example 6 Packed-bed bioreactor using laminin-521 coated macrocarriers. To enable the yields necessary for clinical megakaryocyte and platelet production, it is crucial to transfer the entire differentiation process from small-scale tissue culture plasticware (2D, matrix-dependent) to a 3D scalable solution. Here, we demonstrate that laminin-521-coated PTFE macrocarriers in the shape of 1 mm Raschig rings can provide support for PBG-1 cell differentiation and provide evidence that this macrocarrier material is suitable for use in packed-bed bioreactors, as illustrated in the schematic diagram (Figure 28). PTFE rings were first incubated with 1.25 μg / ml laminin-521 overnight at 4°C on a rocker. Prior to use, the PTFE rings were equilibrated in Essential 8 medium and ROCK inhibitor H1152 in a 6-well plate. Pluripotent PBG-1 iPSCs were harvested using 0.5 mM EDTA, resuspended in Essential 8 medium and H1152, and seeded onto the PTFE rings as clumps. Every 10 minutes, the plates were placed on an orbital shaker at 75 rpm for 30 seconds. After 1 hour, the plates were shaken continuously at 75 rpm overnight. After 24 hours, 90% of the medium was removed and replaced with Phase I medium, with daily medium changes. During Phase I, PBG-1 cells exhibited growth regions inside the Raschig rings (Figure 29), which developed morphological characteristics similar to those seen in 2D cultures (Figure 22). Flow cytometry analysis of these cells revealed a high proportion of hemogenic endothelial cells, with approximately 80% of the cells expressing CD31 and more than half of these cells being double-positive for CD34+ (Figure 31A). Upon switching to Phase II medium and initiating daily half-medium changes, the morphology of the cells changed from generally flat colonies to 3D spheroid-type structures; however, these structures were still attached to the laminin-521 coating inside the ring-shaped macrocarriers (Figure 30). Cells released during phase II had high preMK content as early as day 6+2, with approximately 75% of cells co-expressing CD43 and CD41 (Figure 31B), and purity compared favorably to 2D matrix-dependent cultures (Figure 18A).The cells released on day 6+3 were collected and cultured in ultra-low-attachment plates in Phase III medium for an additional 3 days. Approximately 80% of these cells co-expressed CD61 and CD42b (Figure 31C), indicating efficient MK differentiation. Such macrocarriers are suitable for use as materials for packed-bed bioreactors, which allow for initial differentiation of iPSCs into hemogenic endothelium (i.e., Phase I of directed differentiation) and further differentiation and release of preMK (i.e., Phase II of directed differentiation) within the same vessel (Figure 28). In this design, a packed-bed bioreactor is set up using laminin-521-coated macrocarriers freshly seeded with pluripotent PBG-1 iPSCs. The packed bed is then exposed to continuous medium flow to allow Phase I differentiation into hemogenic endothelium. After permeating the packed bed, the medium circulates through the conditioning chamber, where fresh medium components are added, oxygen / CO2 concentrations are adjusted by sparging or other means, and the medium is then recirculated to the cells. Upon completion of Phase I, the medium is switched to allow Phase II differentiation and the production and release of preMKs. Appropriately sized and shaped macrocarrier supports, such as 1 mm Raschig rings, allow sufficient medium flow and channel width to allow released cells to permeate through the packed bed and out of the reactor for collection and cryopreservation. This design reduces shear forces experienced by the cells, allows efficient medium use due to its perfusion-based design, and allows for the continuous collection of preMKs as they are released.

[0131] Example 7 Self-aggregating iPSC-derived spheroids in a stirred tank bioreactor Another example of a scalable 3D solution involves performing differentiation using self-aggregating spheroids suspended in a stirred or shaking ultra-low attachment vessel (Figure 32). In this example, pluripotent PBG-1 iPSCs were dissociated into single cells using TrypLE and resuspended at 0.5-1 million cells per ml in pluripotency maintenance medium (e.g., Essential 8, Nutristem, StemFlex, other similar media, or a combination thereof) and H1152 or other ROCK inhibitors. The cells were incubated at 37°C, 5% CO2, 20% O2 in a 6-well ultra-low attachment plate on an orbital shaker at 90 rpm or in a spinner flask with constant agitation (90 rpm for a 50 ml volume in a 125 ml spinner flask). Within 24 hours in both systems, PBG-1 cells self-aggregated to form spheroids approximately 50–150 μm in diameter (Figure 33A; see also Figure 11A for a similar example in a different vessel). Agitation was then stopped, and the spheroids were allowed to settle to the bottom of the vessel (approximately 5 minutes). 50–100% of the medium was then replaced with Phase I differentiation medium to promote differentiation into hemogenic endothelium, and agitation was resumed along with incubation under hypoxic conditions (37°C, 5% CO2, 5% O2). Medium changes were repeated daily for a total of 6 days (4 days at 37°C, 5% CO2, 5% O2, followed by 2 days at 37°C, 5% CO2, 20% O2). During this period, the spheroids grew larger, developing characteristic structures and shapes by day 6 (Figure 33A). A sample of these spheroids on day 6 was dissociated and evaluated by flow cytometry. Approximately 44% of the cells were found to express the hemogenic endothelial markers CD31 and CD34 (Figure 33B), with purity comparable to that of 2D matrix-dependent cultures (Figure 16B). To transition to phase II, agitation was stopped and the spheroids were allowed to settle to the bottom of the vessel (approximately 5 minutes). Then, 50–100% of the medium was replaced with phase II differentiation medium to promote differentiation and release of the suspended cells (Figure 34A). Suspended cells were collected and partial medium changes were performed daily thereafter. To do this, agitation was stopped and the hemogenic endothelial spheroids were allowed to settle to the bottom of the vessel (approximately 5 minutes).Approximately 80% of the medium (along with the suspended cells) was collected and centrifuged. Half the working volume of fresh Phase II differentiation medium was added to the spheroids along with a sufficient volume of conditioned medium (i.e., the supernatant after centrifugation) to return to the original working volume. The remaining supernatant was then discarded, and a portion of the cell pellet was used for FACS analysis (Figure 34B), while the remainder was cryopreserved or transferred to Phase III for maturation into mature MKs. Flow cytometry analysis of suspended cells revealed that the majority of cells released between days 6+2 and 6+6 coexpressed the preMK markers CD43 and CD41 (Figures 34B, 34C). Overall preMK purity and yield from 3D self-aggregating spheroid cultures (Figures 34C, 34D) compared favorably with those from 2D cultures (Figures 18A, 19A). Upon transition to static phase III culture, preMKs from 3D self-aggregating spheroid cultures yielded MK purities similar to those from 2D culture systems (Figures 35A-35C). Furthermore, phase III differentiation cultures generated from 3D self-aggregating spheroid cultures contained cells that dramatically increased in size and were capable of generating platelet precursors (Figure 36), consistent with their bona fide megakaryocyte identity.

[0132] Example 8 Detailed characterization of PBG1 iPSC-derived megakaryocytes Megakaryocytes generated using the methods described herein exhibit many features associated with functional, mature MKs, including the MK-specific protein beta-1-tubulin (Figure 37), as well as proteins associated with alpha granules (PF4 and VWF, Figures 38A-38F) and dense granules (LAMP1 and serotonin, Figures 39A-39F) when imaged by immunofluorescence microscopy. Electron microscopy images of PBG1-derived MKs reveal characteristic ultrastructural features, including multivesicular bodies, glycogen granules, and invaginated membrane systems (Figures 40A-40D). Gene expression analysis revealed downregulation of pluripotency genes such as OCT4 (Figure 41A) and upregulation of megakaryocyte lineage genes such as NFE2 (Figure 41B). Similar analysis was performed on a panel of relevant genes, and the results of this analysis are consistent with the loss of a pluripotent stem cell signature and the acquisition of a megakaryocyte signature (Figure 41C).

[0133] Bone marrow CD34 + Cells, peripheral blood CD34 + cells, or cord blood CD34 + Compared to primary megakaryocytes (natural products) derived from PBG1 cells, the PBG1-derived MKs found to be PBG1 iPSC-derived MKs had a similar average size (Figures 42A-42C), but nevertheless, CD34 +The PBG1 hiPSC-derived megakaryocytes had a characteristic hypoploidy distribution (Figures 43A-43B) compared with primary megakaryocytes (natural product) derived from bone marrow, peripheral blood, or umbilical cord blood stem cells (Figures 42C, 43B). The PBG1 hiPSC-derived megakaryocytes also had a characteristic growth factor, cytokine, and chemokine expression profile similar to that present in human platelets, including the presence of numerous factors not previously reported in megakaryocytes (Figure 44). To prepare the data, hiPSC-MK cells at 25 million cells / mL in 1x PBS were lysed by freezing the cells overnight at -80°C and then thawing at 37°C. This freeze / thaw cycle was repeated four times. The resulting suspension was filtered using a 0.22 μm syringe filter. The lysates were tested for a select panel of growth factors, cytokines, and chemokines using multiplexed laser bead technology (Eve Technologies). Data were corrected for background (PBS, treated identically to hiPSC-MK) and then compared to commercially available human platelet lysate (HPL), fresh MK differentiation medium (used at the final stage of differentiation), and conditioned medium, i.e., MK differentiation medium removed from hiPSC-MK before lysis. While a strong overlap was observed between hiPSC-MK and HPL, several proteins not previously described in megakaryocytes or platelets were also measured in hiPSC-MK (indicated by "").

[0134] The results described herein demonstrate a robust process for generating clinical-grade human iPSC-derived megakaryocytes, which can be isolated and enriched for further characterization or use in downstream applications, such as the generation of human platelets (Figures 45A-45C).

[0135] Other embodiments As described herein, the present disclosure features compositions and methods for producing megakaryocyte progenitor cells and megakaryocytes. In one aspect, the present disclosure provides megakaryocytes or megakaryocyte progenitor cells differentiated from clinical-grade hiPSC cells or cell lines. In some embodiments, the present disclosure provides an isolated population of cells comprising megakaryocytes or megakaryocyte progenitor cells according to any aspect delineated herein. In some embodiments, the present disclosure provides a composition comprising megakaryocytes or megakaryocyte progenitor cells according to any aspect delineated herein.

[0136] In some embodiments, the present disclosure provides a composition or pharmaceutical composition comprising a lysate of megakaryocytes according to any aspect delineated herein. In some embodiments, the present disclosure provides a composition or pharmaceutical composition comprising a lysate of platelets generated from megakaryocytes according to any aspect delineated herein. In some embodiments, the present disclosure provides a method of producing megakaryocytes, the method comprising differentiating clinical-grade hiPSC cells or cell lines. In various embodiments of any aspect delineated herein, the megakaryocytes are CD42b + , CD61 + , and DNA + In various embodiments of any aspect delineated herein, the megakaryocytes have a diameter of about 10-30 μm. In certain embodiments, the megakaryocytes have a diameter of about 10-20 μm. In various embodiments of any aspect delineated herein, the megakaryocytes have a ploidy of at least 4N, 8N, or 16N. In various embodiments of any aspect delineated herein, the megakaryocyte progenitor cells are CD14 - , CD41 + , and CD43 +In various embodiments of any aspect delineated herein, megakaryocyte progenitor cells can be continuously produced until at least day 10 after initiation of differentiation from hemogenic endothelial progenitor cells (i.e., Phase II, days 6+10). In various embodiments of any aspect delineated herein, megakaryocyte progenitor cells can be continuously produced until at least day 17 after initiation of differentiation from hemogenic endothelial progenitor cells (i.e., Phase II, days 6+17). In various embodiments of any aspect delineated herein, the clinical-grade hiPSC cells or cell lines are selected from the group consisting of PBG1, PBG2, and PBG3.

[0137] In various embodiments of any aspect delineated herein, the isolated cell population or composition contains platelets generated from megakaryocytes of the present disclosure. In various embodiments of any aspect delineated herein, the isolated cell population or composition contains CD41 + CD61 + CD42b among cells + In various embodiments of any aspect delineated herein, the isolated cell population or composition contains at least 50% megakaryocytes with a ploidy of 4N or greater. In certain embodiments, at least 50% of the megakaryocytes have a ploidy of 4N to 16N. In various embodiments of any aspect delineated herein, the isolated cell population or composition contains megakaryocytes with an average ploidy of 4N or greater.

[0138] From the foregoing description, it will be apparent that variations and modifications can be made to the disclosure described herein to adapt it to various usages and conditions, and such embodiments still fall within the scope of the following claims.

[0139] The recitation of a listing of elements in any definition of a variable herein includes that definition of the variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or combination with any other embodiment or portion thereof.

[0140] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference. The present invention provides, for example, the following items. (Item 1) 1. A method for producing megakaryocytes, comprising: Expanding pluripotent stem cells under low-adherence or non-adherence conditions and under agitation, wherein the expanded pluripotent stem cells form self-aggregating spheroids; differentiating the pluripotent cells into hemogenic endothelial cells in a first culture medium; differentiating the hemogenic endothelial cells into megakaryocyte progenitor cells in a second culture medium; A method comprising: (Item 2) Item 10. The method according to item 1, wherein the step of differentiating the pluripotent cells into hemogenic endothelial cells is carried out on a matrix under adhesive conditions. (Item 3) 3. The method of claim 2, wherein the matrix comprises laminin. (Item 4) 3. The method of claim 2, wherein the matrix is ​​attached to a two-dimensional surface. (Item 5) 3. The method of claim 2, wherein the matrix is ​​attached to a three-dimensional structure. (Item 6) 2. The method of claim 1, wherein the step of differentiating the pluripotent cells into hemogenic endothelial cells is carried out under low-adherence or non-adherence conditions to allow self-aggregation of the hemogenic endothelial cells. (Item 7) 2. The method of claim 1, wherein the first culture medium comprises one or more of bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF). (Item 8) 8. The method of claim 7, wherein the first culture medium further comprises a WNT modulator. (Item 9) 2. The method of claim 1, wherein the second culture medium comprises one or more of stem cell factor (SCF), thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (Flt3-L), interleukin-3 (IL-3), interleukin-6 (IL-6), and heparin. (Item 10) Item 2. The method according to item 1, wherein the pluripotent stem cells are human induced pluripotent stem cells. (Item 11) Item 14. The method of item 1, further comprising the step of collecting and dissociating the expanded pluripotent stem cells. (Item 12) 2. The method of claim 1, further comprising the step of seeding the megakaryocyte progenitor cells on a non-adherent surface in a culture medium prior to the step of differentiating the megakaryocyte progenitor cells into megakaryocytes. (Item 13) 13. The method of any one of items 1 to 12, further comprising differentiating the megakaryocyte progenitor cells into megakaryocytes in a third culture medium. (Item 14) 14. The method of claim 13, wherein the third culture medium comprises one or more of stem cell factor (SCF), thrombopoietin (TPO), interleukin-6 (IL-6), interleukin-9 (IL-9), and heparin. (Item 15) 1. A method for producing megakaryocytes, comprising: differentiating the pluripotent cells into hemogenic endothelial cells in a first culture medium; differentiating the hemogenic endothelial cells into megakaryocyte progenitor cells in a second culture medium; Including, The method, wherein at least one of the steps of differentiating the pluripotent cells and differentiating the hemogenic endothelial cells is performed on a matrix-coated three-dimensional structure. (Item 16) 16. The method of claim 15, wherein the three-dimensional structure is a microcarrier. (Item 17) 16. The method of claim 15, wherein the three-dimensional structure is a microcarrier. (Item 18) Item 16. The method of item 15, wherein the matrix comprises laminin. (Item 19) 16. The method of item 15, wherein the first culture medium comprises one or more of bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF). (Item 20) 16. The method of claim 15, wherein the first culture medium further comprises a WNT modulator. (Item 21) 16. The method of item 15, wherein the second culture medium comprises one or more of stem cell factor (SCF), thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (Flt3-L), interleukin-3 (IL-3), interleukin-6 (IL-6), and heparin. (Item 22) Item 16. The method according to item 15, wherein the pluripotent stem cells are human induced pluripotent stem cells. (Item 23) Item 16. The method of item 15, further comprising expanding pluripotent stem cells in the matrix-coated three-dimensional structure. (Item 24) 24. The method of any one of items 15 to 23, further comprising differentiating the megakaryocyte progenitor cells into megakaryocytes in a third culture medium. (Item 25) 25. The method of claim 24, wherein the third culture medium comprises one or more of stem cell factor (SCF), thrombopoietin (TPO), interleukin-6 (IL-6), interleukin-9 (IL-9), and heparin. (Item 26) 1. A method for producing megakaryocytes, comprising: differentiating the pluripotent cells into hemogenic endothelial cells in a first culture medium; differentiating the hemogenic endothelial cells into megakaryocyte progenitor cells in a second culture medium; Including, The method, wherein at least one of the steps of differentiating the pluripotent cells and differentiating the hemogenic endothelial cells is performed under low-adherence or non-adherence conditions to allow cells to self-aggregate. (Item 27) 27. The method of claim 26, wherein the first culture medium comprises one or more of bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF). (Item 28) 28. The method of claim 27, wherein the first culture medium further comprises a WNT modulator. (Item 29) 27. The method of item 26, wherein the second culture medium comprises one or more of stem cell factor (SCF), thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (Flt3-L), interleukin-3 (IL-3), interleukin-6 (IL-6), and heparin. (Item 30) 27. The method of item 26, wherein the pluripotent stem cells are human induced pluripotent stem cells. (Item 31) 31. The method of any one of items 26 to 30, further comprising differentiating the megakaryocyte progenitor cells into megakaryocytes in a third culture medium. (Item 32) 32. The method of claim 31, wherein the third culture medium comprises one or more of stem cell factor (SCF), thrombopoietin (TPO), interleukin-6 (IL-6), interleukin-9 (IL-9), and heparin. (Item 33) 31. A composition comprising megakaryocyte progenitor cells produced by the method of any one of items 1 to 12, 15 to 23, or 26 to 30. (Item 34) 31. A composition comprising a lysate of megakaryocyte progenitor cells produced by the method of any one of items 1 to 12, 15 to 23, or 26 to 30. (Item 35) 33. A composition comprising megakaryocytes or a lysate of megakaryocytes produced by any one of the methods of items 13, 14, 24, 25, 31 or 32. (Item 36) The megakaryocytes are CD42b + , CD61 + , and DNA + Item 36. The composition according to item 35, wherein (Item 37) 14. A composition comprising megakaryocytes or a megakaryocyte lysate produced by the method of item 13. (Item 38) The megakaryocytes are CD42b + , CD61 + , and DNA + Item 38. The composition according to item 37, wherein (Item 39) 15. A composition comprising megakaryocytes or a megakaryocyte lysate produced by the method of item 14. (Item 40) The megakaryocytes are CD42b + , CD61 + , and DNA + Item 39. The composition according to item 39, wherein (Item 41) 25. A composition comprising megakaryocytes or a megakaryocyte lysate produced by the method of item 24. (Item 42) The megakaryocytes are CD42b + , CD61+ , and DNA + Item 42. The composition according to item 41, wherein (Item 43) 26. A composition comprising megakaryocytes or a megakaryocyte lysate produced by the method of item 25. (Item 44) The megakaryocytes are CD42b + , CD61 + , and DNA + Item 44. The composition according to item 43, wherein (Item 45) 32. A composition comprising megakaryocytes or a megakaryocyte lysate produced by the method of claim 31. (Item 46) The megakaryocytes are CD42b + , CD61 + , and DNA + Item 46. The composition according to item 45, wherein (Item 47) 33. A composition comprising megakaryocytes or a megakaryocyte lysate produced by the method of item 32. (Item 48) The megakaryocytes are CD42b + , CD61 + , and DNA + Item 48. The composition according to item 47, wherein

Claims

[Claim 1] The invention as described in the drawings.

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