Method for production of megakaryocytes

Abstract

Methods are provided for the production of megakaryocytic progenitors (premks) and megakaryocytes (mks) from stem cells. This disclosure further provides compositions comprising premks and mks and their lysates, as well as methods for using premks, mks, their lysates and compositions thereof.

Description

1 / 97 METHOD FOR PRODUCING MEGAKARYOCYTES GOVERNMENT STATEMENT OF SUPPORT

[001] This work was funded by the following grants from the National Institutes of Health, Grants No: 1R44HL131050-01, 1R43AI125134-01A1 and 1SB1HL137591-01. The The government has certain rights to the invention. RELATED ORDERS

[002] This application claims the benefit and priority of U.S. Provisional Application Serial No. 62 / 614117, filed January 5, 2018, the entirety of which is incorporated herein by reference. FIELD

[003] This disclosure relates to methods for producing megakaryocytic progenitors and megakaryocytes, compositions of megakaryocytic progenitors and megakaryocytes, and their uses. FUNDAMENTALS

[004] Platelets are blood cells responsible for clot formation and repair of blood vessels at sites of active bleeding. Physiologically, platelets are produced in the bone marrow by stem cells called megakaryocytes (MKs), which comprise <0.1% of the cells in the bone marrow. Mature MKs reside outside sinusoidal blood vessels in the bone marrow and extend long distances. Petition 870260066603, dated 06 / 07 / 2026, page 16 / 211 2 / 97 structures called proplatelets for circulation. Proplatelets function like assembly lines for platelet production and sequentially release platelets from their ends.

[005] MKs are produced through a multi-step differentiation process from hematopoietic stem cells in the bone marrow. 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. After further differentiation, including cell enlargement, increased DNA content, endomitosis, and granule formation, mature MKs are produced. MKs convert their cell mass into proplatelet extensions to produce / release anucleated platelets.

[006] Low platelet count is a significant consequence of several diseases and therapies, including cancer treatment, transplantation, and surgery, for which platelets are a critical first-line therapy to prevent mortality due to uncontrolled bleeding. Platelet units (3 x 10⁶ x 1 platelets per 200-400 mL) are derived exclusively from voluntary human donors and must be stored at the temperature Petition 870260066603, dated 06 / 07 / 2026, page 17 / 211 3 / 97 environment to prevent irreversible activation. However, at this temperature, there is a risk of bacterial growth, limiting the shelf life of a platelet unit to 5 days, 2 of which are consumed for pathogen screening and 1 for transport. Consequently, blood centers typically have no more than 1.5 days of platelet inventory available for transfusion, which is rapidly depleted during emergencies. Platelet donor shortages can occur, especially during critical times. The increasing demand for civilian use alone exceeds supply by ~20%, and reserve stocks are quickly exhausted in emergencies. Furthermore, the wide functional variability between units and donors leads to overtransfusion to ensure effective bleeding control. Consequently, megakaryocytes, which provide a source of platelets, and new improved methods of megakaryocyte generation are urgently needed. SUMMARY

[007] This disclosure provides methods for the production of megakaryocytic progenitors (preMKs) and megakaryocytes (MKs) from stem cells. This disclosure also provides compositions comprising preMKs and MKs and their lysates, as well as methods for using preMKs, MKs, their lysates and compositions thereof.

[008] In some embodiments, the present Petition 870260066603, dated 06 / 07 / 2026, page 18 / 211 Disclosure 4 / 97 provides a method for the production of megakaryocytes comprising: expanding pluripotent stem cells under low-adherent or non-adherent conditions and under agitation wherein the expanded pluripotent stem cells form self-aggregating spheroids; differentiating the pluripotent cells in a first culture medium into hemogenic endothelial cells; differentiating the hemogenic endothelial cells in a second culture medium into megakaryocytic progenitors. The differentiation of pluripotent cells into hemogenic endothelial cells can be performed under adherent conditions in a matrix. In some embodiments, the differentiation of pluripotent cells into hemogenic endothelial cells is performed under low-adherent or non-adherent conditions to allow the hemogenic endothelial cells to self-aggregate.

[009] In some embodiments, the present disclosure provides a method for producing megakaryocytes comprising: differentiating 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, wherein at least one of the differentiations of pluripotent cells and differentiation of hemogenic endothelial cells is carried out in a matrix-coated three-dimensional structure. The three-dimensional structure may be a Petition 870260066603, dated 06 / 07 / 2026, page 19 / 211 5 / 97 microcarrier or a microcarrier.

[0010] In some embodiments, the present disclosure provides a method for producing megakaryocytes comprising: differentiating 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, wherein at least one of the differentiations of pluripotent cells and differentiation of hemogenic endothelial cells is carried out under conditions of low adhesion or non-adhesion to allow the cells to self-aggregate.

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

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

[0013] In some embodiments, the methods Petition 870260066603, dated 06 / 07 / 2026, page 20 / 211 6 / 97 present may also include a step of expansion of pluripotent stem cells in the three-dimensional matrix-coated structure.

[0014] In some embodiments, the present methods may further include a step of differentiation of megakaryocytic progenitors in a third culture medium into megakaryocytes. The third medium may comprise one or more stem cell factors (SCF), thrombopoietin (TPO), interleukin-6 (IL-6), interleukin-9 (IL-9), and heparin.

[0015] In some embodiments, the present disclosure provides megakaryocytic parent compositions or megakaryocytic parent 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, these megakaryocytes are CD42b+, CD61+, and DNA+.

[0017] Other features and advantages of the present disclosure will be evident from the following description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present disclosure will be described in the detailed description that follows, with reference to the plurality of Petition 870260066603, dated 06 / 07 / 2026, page 21 / 211 7 / 97 drawings noted by means of non-limiting examples of exemplary embodiments, in which similar reference numbers represent similar parts along the various views of the drawings and in which:

[0019] FIG. 1 shows a general scheme for scalable differentiation of megakaryocytic progenitors (preMKs), megakaryocytes (MKs) and platelets (PLTs) from an iPSC cell line.

[0020] FIG. 2 represents an example of a directed differentiation protocol of a pluripotent stem cell into a megakaryocyte in a 2D matrix-dependent system, such as a cell culture plate or flask.

[0021] FIG. 3A and FIG. 3B represent the pluripotency of 3 exemplary clinical-grade hiPSC lines (hereinafter referred to as PBG1, PBG2, PBG3). FIG. 3A shows low-magnification phase-contrast images of PBG1, PBG2, and PBG3 iPSCs forming characteristic growth areas when cultured in Vitronectin matrix with Essential 8 medium. FIG. 3B shows higher-magnification images of PBG1, PBG2, and PBG3 iPSCs immunostained for the pluripotency factors Oct4 and Nanog, and contrasted with a nuclear dye.

[0022] FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D represent iPSC PBG1, PBG2, and PBG3 cultures progressing through phases of directed cell differentiation. Petition 870260066603, dated 06 / 07 / 2026, p. 22 / 211 8 / 97 pluripotent stem cells to mature megakaryocytes. FIG. 4A represents a general schematic of the timeline of the directed differentiation process. FIG. 4B shows actual images of PBG1 cultures during Phase 0 (day 0), Phase I (day 2 and day 5), Phase II (day 6 + 7), and Phase III (day +2 and +4). FIG. 4C shows actual images of PBG2 cultures during Phase 0 (day 0), Phase I (day 2 and day 5), Phase II (day 6 + 7), and Phase III (day +2 and +4). FIG. 4D shows actual images of PBG3 cultures during Phase 0 (day 0), Phase I (day 2 and day 5), Phase II (day 6 + 7), and Phase III (day +2 and +4).

[0023] FIG. 5 is a graph representing the average CD31+ differentiation efficiency at the end of Phase I for targeted differentiation of the iPSC lines PBG1, PBG2 and PBG3.

[0024] FIG. 6A, FIG. 6B and FIG. 6C show the purity and yields of megakaryocytic progenitors (CD43+CD41+) generated during Phase II targeted differentiation of the iPSC lines PBG1, PBG2 and PBG3. FIG. 6A represents representative CD41 / CD43 flow cytometry data for suspension cells harvested from Phase II cultures on day 6+6 (from left to right: PBG1, PBG2, PBG3). FIG. 6B is a graph showing daily output measurements of CD41+CD43+ cells (megakaryocytic progenitor) released in suspension during Phase II differentiation cultures representative of the iPSC lines PBG1, PBG2 and PBG3. The production of CD41+CD43+ megakaryocytic progenitors in Petition 870260066603, dated 06 / 07 / 2026, page 23 / 211 9 / 97 PBG1 and PBG2 differentiation cultures were measured up to Day 6+17, while CD41+CD43+ cell production in PBG3 differentiation cultures stopped on Day 6+8. FIG. 6C represents the cumulative yield of CD41+CD43+ cells during Phase II differentiation cultures of grade hiPSC lines, shown as the number of CD41+CD43+ cells / well.

[0025] FIG. 7A, FIG. 7B, and FIG. 7C represent Phase III, the final phase of the targeted differentiation protocol, which ultimately generates mature megakaryocytes. FIG. 7A represents the maturation status of PBG1, PBG2, and PBG3-derived cells over time in Phase III cultures, as measured by the proportion of CD41+ megakaryocytic lineage cells that also express the mature megakaryocyte marker CD42b. FIGs. 6B and 6C represent light microscopy images of hiPSC-derived megakaryocytes on day 4 of Phase III that have been differentiated from PBG1 and PBG2, respectively. Examples of proplatelet extensions are indicated with arrows.

[0026] FIGS. 8A, 8B, 8C, 8D, and 8E represent the characterization of mature megakaryocytes derived from the differentiation of PBG1, PBG2, and PBG3 hiPSCs. FIG. 8A represents Phase III cells derived from PBG1 that were immunostained for p1-tubulin, and the nuclei were visualized by nucleic acid staining (top panel). Megakaryocytes derived from PBG1 were also Petition 870260066603, dated 06 / 07 / 2026, p. 24 / 211 10 / 97 analyzed by electron microscopy (lower panel). FIG. 8B represents Phase III cells derived by differentiation from PBG2 hiPSCs. Megakaryocytes derived from PBG2 were immunostained for p1-tubulin, and the nuclei were visualized by nucleic acid staining (upper panel). Megakaryocytes derived from PBG2 were also analyzed by electron microscopy (lower panel). FIG. 8C represents Phase III cells derived by differentiation from PBG3 hiPSCs. Megakaryocytes derived from PBG3 were immunostained for p1-tubulin, and the nuclei were visualized by nucleic acid staining (upper panel). Megakaryocytes derived from PBG3 were not analyzed by electron microscopy (lower panel). FIG. 8D represents the proportion of Phase III cells derived from PBG1, PBG2, and PBG3 iPSCs that stained positive for intracellular von Willebrand factor.8E represents the proportion of Phase III cells derived from the iPSC PBG1, PBG2, and PBG3 lines that stained positive for intracellular platelet factor 4.

[0027] FIGS. 9A, 9B and 9C represent the expansion of pluripotent PBG1 cells in recombinant vitronectin using various growth media. FIG. 9A shows the growth of PBG1 in Essential 8 medium. FIG. 9B shows the growth of PBG1 in StemFlex medium. FIG. 9C shows the growth of PBG1 in Nutristem XF medium. Petition 870260066603, dated 06 / 07 / 2026, page 25 / 211 11 / 97

[0028] FIGS. 10A, 10B, and 10C represent flow cytometry data evaluating the expression of the pluripotency markers Tra-1-60, SSEA5, and the differentiation marker SSEA1 in PBG1 cells expanded in recombinant vitronectin using various growth media. FIG. 10A shows pluripotency marker data from PBG1 cells expanded in Essential 8 medium. FIG. 10B shows pluripotency marker data from PBG1 cells expanded in StemFlex medium. FIG. 10C shows pluripotency marker data from PBG1 cells expanded in Nutristem XF medium.

[0029] FIGS. 11A, 11B, and 11C depict the expansion of PBPS1 iPSCs in self-aggregating spheroid cultures in a 3D shake tank (without a matrix). FIG. 11A shows microscopic images of PBG1 spheroids over time in culture. FIG. 11B depicts the increase in cell density in PBG1 spheroid cultures over time. FIG. 11C depicts the average size of the PBG1 spheroid over time in 3D culture.

[0030] FIGS. 12A and 12B represent flow cytometry data evaluating the expression of the pluripotency markers Tra-1-60, SSEA5, and the differentiation marker SSEA1 in PBG1 cells expanded in self-aggregating spheroid cultures in a 3D shake tank (without matrix). FIG. 12A shows data for the marker of Petition 870260066603, dated 06 / 07 / 2026, p. 26 / 211 Figure 12A shows pluripotency data for PBG1 cells after a single 7-day expansion in a 3D shake tank.

[0031] FIGS. 13A and 13B represent PBG1 iPSCs immunostained for the pluripotency factors Oct 4 and Nanog and counterstained with a nuclear dye. FIG. 13A represents a portion of a 2D colony of PBG-1 iPSCs grown in vitronectin. FIG. 13B represents a spheroid of PBG-1 iPSCs grown under agitated 3D conditions (without matrix).

[0032] FIG. 14 represents the karyotype analysis of a metaphase chromosome spread from PBG1 iPSCs grown by 4 consecutive 6-7 day expansions in a 3D shaking tank, demonstrating a normal karyotype after 4 rounds of 3D passage.

[0033] FIG. 15 shows the morphological changes that occur over 6 days of differentiation from Phase I iPSC PBPS to hemogenic endothelium in collagen matrix IV in a 2D culture vessel.

[0034] FIGS. 16A and 16B represent representative Phase I differentiation data for PBG1-derived cells. FIG. 16A represents a representative cytometric analysis of the flow of PBG1-derived cells on day 6 of differentiation. Hemogenic endothelial cells are identified through the expression of Petition 870260066603, dated 06 / 07 / 2026, page 27 / 211 13 / 97 cell surface of CD31 and CD34. FIG. 16B represents the mean and range of Phase I (day 6) differentiation efficiencies in 41 PBG1-independent directed differentiations.

[0035] FIGS. 17A, 17B, and 17C represent representative Phase II data from PBG1 differentiation cultures. FIG. 17A shows a Phase II culture on day 6+6, with the hemogenic endothelial monolayer (HE) in the background and megakaryocytic progenitors (preMKs) being released from the monolayer into suspension. FIG. 17B shows the flow cytometric analysis of Phase II cells in suspension, identifying CD43+ hematopoietic progenitor cells. FIG. 17C shows the flow cytometric analysis of CD43+ hematopoietic cells, identifying CD43+ CD41+ CD14- megakaryocytic progenitors (preMKs). Contaminating CD43+CD14+ myeloid progenitors are also identified in this analysis.

[0036] FIGS. 18A and 18B represent average compositional characteristics of Phase II suspension cells. FIG. 18A represents the average daily purity (i.e., the percentage of CD41+CD43+CD14- viable suspension cells) of preMKs released over 10 days of Phase II. FIG. 18B represents the median, quartiles, and ranges of contaminating myeloid progenitors (i.e., the percentage of CD43+CD14+ cells). Petition 870260066603, dated 06 / 07 / 2026, pages 28 / 211 14 / 97 viable suspensions) over 10 days of Phase II. All cultures were initiated with PBG1 cells in a collagen IV matrix in a 2D vessel. The data represent 41 independent differentiations.

[0037] FIG. 19A and FIG. 19B represent yields of released preMKs. FIG. 19A represents the average daily yields of released preMKs (i.e., viable CD41+CD43+CD14) per equivalent of 6 wells (i.e., 2 ml of medium, 9.5 cm2 surface area) during Phase II targeted differentiation cultures initiated with PBG1 iPSCs. FIG. 19B represents the cumulative yields of released preMKs (i.e., viable CD41+CD43+CD14) per equivalent of 6 wells (i.e., 2 ml of medium, 9.5 cm2 surface area) between days 6+4 and 6+8 of Phase II targeted differentiation cultures initiated with PBG1 iPSCs. Each point represents a PBG1-directed independent differentiation culture in the collagen IV matrix within a 2D culture vessel.

[0038] FIGS. 20A, 20B, 20C, and 20D depict MK differentiation and prosthesis production in Phase III. FIG. 20A represents megakaryocytic progenitors derived from PBG1 on Day 1 of Phase III (top panel: high magnification; bottom panel: low magnification). FIG. 20B represents maturing megakaryocytes on Day 2 of Phase III (top panel: high magnification). Petition 870260066603, dated 06 / 07 / 2026, pages 29 / 211 FIG. 20C represents mature megakaryocytes on Day 4 of Phase III (upper panel: high magnification; lower panel: low magnification). FIG. 20D illustrates the spontaneous formation of proplatelets from mature PBG1-derived MKs after 4 days of Phase III culture.

[0039] FIGS. 21A, 21B, and 21C represent a representative flow cytometric analysis of Day 3 Phase III cultures initiated from PBG1 iPSCs. FIG. 21A identifies the CD61+ (megakaryocytic) fraction of Phase III cells. FIG. 21B shows the flow cytometric analysis of CD61+ megakaryocytic cells, identifying mature CD42a+CD42b+ MKs. Apoptotic CD42a+CD42b- cells can also be identified in this analysis. FIG. 21C represents the 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+.

[0040] FIGS. 22A and 22B show the use of Laminin 521 and Collagen IV in Phase I of targeted differentiation of PBG1 cells. FIG. 22A shows the progression of Phase I differentiation at 4.2 ug / cm2 of human collagen IV. FIG. 22B shows the progression of Phase I differentiation at 0.13 ug / cm2 of recombinant human laminin 521.

[0041] Figures 23A, 23B and 23C describe the use Petition 870260066603, dated 06 / 07 / 2026, page 30 / 211 16 / 97 of recombinant Laminin 521 to support the production and release of megakaryocytic progenitors in Phase II targeted differentiation of PBG1 cells. FIG. 23A represents representative flow cytometry data from Phase II PBG1 differentiation cultures using a support matrix of 4.2 µg / cm² of human Collagen IV. FIG. 23B represents representative flow cytometry data from Phase II PBG1 differentiation cultures using a support matrix of 0.13 µg / cm² recombinant human Laminin 521. 23C represents the cumulative yields of preMKs released (i.e., viable CD41+CD43+CD14) per equivalent of 6 wells (i.e., 2 ml of medium, 9.5 cm2 surface area) between days 6+4 and 6+8 of Phase II targeted differentiation cultures initiated with PBG1 iPSCs, using a support matrix of 4.2 μg / cm2 of human Collagen IV or 0.13 μg / cm2 of recombinant human Laminin 521.

[0042] FIGS. 24A and 24B show the production of proplatelets from differentiated MKs from preMKs generated from Laminin 521 cultures. Phase III cultures (day 6+6+3) initiated with preMKs from collagen IV cultures are shown in FIG. 24A, and Phase III (day 6+6+3) initiated with preMKs from Laminin 521 cultures are shown in FIG. 24B. Red arrows indicate examples of proplatelets. Petition 870260066603, dated 06 / 07 / 2026, page 31 / 211 17 / 97

[0043] FIGS. 25A and 25B show flow cytometry subset breaks of Phase III (Day 6+6+3) cultures. FIG. 25A shows a flow cytometry subset break of Phase III (Day 6+6+3) cultures initiated with preMKs from collagen IV cultures. FIG. 25B shows a flow cytometry subset break 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+.

[0044] FIGS. 26A, 26B, and 26C represent immunofluorescence microscopy images of Phase I Day 6 cultures on Laminin 521. FIG. 26A represents a control culture without WNT agonist. FIG. 26B represents a culture in which 0.6 µM of the WNT agonist CHIR98014 was added to the differentiation culture during the first 48 hours of Phase I. FIG. 26C represents a culture in which 6 µM of the WNT agonist CHIR99021 was added to the differentiation culture during the first 48 hours of Phase I.

[0045] FIGS. 27A and 27B represent immunofluorescence microscopy images of Day 6+4 Phase II cultures on Laminin 521. FIG. 27A represents a control culture without WNT agonist. FIG. 27B represents a Petition 870260066603, dated 06 / 07 / 2026, p. 32 / 211 18 / 97 culture in which 0.6uM of the WNT agonist CHIR98014 was added within the first 48 hours of Phase I.

[0046] FIG. 28 is a schematic representing an example of a targeted differentiation protocol of PBG1 into megakaryocytic progenitors using a packed bed bioreactor strategy, a 3D, matrix-dependent method. In the embodiment described here, Laminin 521-coated Raschig rings made of PTFE are used as macrocarriers to compose the packed bed.

[0047] FIG. 29 represents the Phase I differentiation of PBG-1 iPSCs in Rachig rings coated with Laminin 521 on day 3 and day 6.

[0048] FIG. 30 represents the Phase II differentiation of PBG-1 iPSCs in Rachig rings coated with Laminin 521 on day 6+0 and day 6+4.

[0049] FIGS. 31A, 31B, and 31C represent flow cytometric data of PBG1 differentiation phases, efficiently processed on Rachig ring substrate. FIG. 31A represents Phase I on day 6, with flow cytometric staining for the hemogenic endothelial markers CD31 and CD34. FIG. 31B represents Phase II on day 6+2, with flow cytometric staining for the megakaryocytic progenitor markers CD43 and CD41. FIG. 31C represents Phase III on day 6+3+3, with flow cytometric staining for CD61 and CD42b. Petition 870260066603, dated 06 / 07 / 2026, page 33 / 211 19 / 97

[0050] FIG. 32 is a schematic of an example of a matrix-independent 3D method of targeted differentiation using self-aggregating iPSC-derived spheroids in a stirring tank.

[0051] FIGS. 33A and 33B represent Phase 0 and Phase I differentiation initiated with self-aggregating spheroids of PBG1 iPSCs. FIG. 33A shows a series of micrographs, beginning with single-cell dissociated PBG1 cells on day -1, self-aggregating PBG1 iPSC spheroids on day 0, partially differentiated spheroids on day 3, and fully differentiated spheroids containing hemogenic endothelial cells on day 6. FIG. 33B represents flow cytometric data on day 6, showing successful CD31+CD34+ hemogenic endothelial differentiation using this approach.

[0052] FIGS. 34A, 34B, 34C, and 34D depict Phase II in a targeted differentiation initiated with self-aggregating spheroids of PBG1 iPSCs. FIG. 34A represents self-aggregating spheroids derived from PBG1 on day 6+4 during Phase II of targeted differentiation, with preMKs released from the spheroids in suspension. FIG. 34B represents flow cytometry analysis of harvested suspension cells, stained for the preMK markers CD41 and CD43. FIG. 34C represents the purity of preMK over time in Phase II, in 2 different 3D systems, one vessel Petition 870260066603, dated 06 / 07 / 2026, page 34 / 211 20 / 97 ultra-low adherent vessel in an orbital shaker and a spinning flask. FIG. 34D represents the yields of preMK over time in Phase II in 2 different 3D systems, an ultra-low adherent vessel in an orbital shaker and a spinning flask.

[0053] FIGS. 35A, 35B, and 35C represent Phase III MK differentiation from matrix-independent 3D cultures initiated from self-aggregating spheroids of PBG1 iPSCs. FIG. 35A represents a representative flow cytometric analysis of Day 3 Phase III cultures, identifying the CD61+ (megakaryocytic) fraction of Phase III cells, followed by the identification of mature CD42a+CD42b+ MKs. Apoptotic CD42a+CD42b- cells can also be identified in this analysis. FIG. 35B represents the subset decomposition of a representative Phase III culture. Non-MKs are CD61-, immature MKs are CD61+CD42aCD42b-, apoptotic MKs are CD61+CD42a+CD42b-, and mature MKs are CD61+CD42a+CD42b+. FIG. 35C shows how the mature MK fraction in Phase III cultures on day 3 compares between the 2D (matrix-dependent) and 3D (matrix-independent) approaches.

[0054] FIG. 36 represents extensions of mature MK proplatelets harvested from 3D self-aggregating spheroid differentiation cultures. Examples of proplatelet extensions are indicated with arrows. Petition 870260066603, dated 06 / 07 / 2026, page 35 / 211 21 / 97

[0055] FIG. 37 depicts megakaryocytes derived from PBG1 immunostained for the megakaryocyte-specific p1-tubulin protein. Simultaneously, the nuclei were visualized by nucleic acid staining.

[0056] FIGS. 38A, FIG. 38B, FIG. 38C, FIG. 38D, FIG. Figures 38E and 38F represent PBG1-derived megakaryocytes immunostained for the granule-specific proteins Platelet Factor 4 (PF4), Von Willebrand Factor (VWF), as well as for the megakaryocyte-specific cell surface marker CD61 and nuclei.

[0057] FIGS. 39A, FIG. 39B, FIG. 39C, FIG. 39D, FIG. Figures 39E and 39F represent PBG1-derived megakaryocytes immunostained for the Dense Granule-specific proteins LAMP1 and serotonin, as well as for the megakaryocyte-specific cell surface marker CD61 and nuclei.

[0058] FIGS. 40A, 40B, 40C, and 40D are electron microscopy images showing a PBG1-derived megakaryocyte. FIG. 40A is an electron microscopy image showing PBG1-derived megakaryocyte-producing microparticles (see arrows for examples). FIG. 40B is an electron microscopy image showing a PBG1-derived megakaryocyte and multivesicular bodies (arrows; enlarged at inset). FIG. 40C is an electron microscopy image showing a PBG1-derived megakaryocyte characterized by nuclei of Petition 870260066603, dated 06 / 07 / 2026, page 36 / 211 22 / 97 multiple lobes, glycogen granules, alpha granules, and an invaginated membrane system. FIG. 40D is an electron microscopy image showing the endoplasmic reticulum and mitochondria of a PBG1-derived megakaryocyte.

[0059] Figures 41A, 41B, and 41C illustrate characteristic gene expression changes that occur during the course of directed differentiation of PBG1 cells into megakaryocytes. For all expression analyses, expression in pluripotent PBG1 cells was set to 1, and all other expression values ​​are presented in relative terms. Figure 41A illustrates the relative gene expression of Oct4, a gene associated with pluripotency, in pluripotent PBG1 cells, day 6 cells (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 critical for megakaryocyte maturation, in pluripotent PBG1 cells, day 6 cells (end of phase I), days 6+4 and 6+5 (phase II), and days 6+5+1 to 6+5+4 (phase III). Similar analyses were performed on a panel of relevant genes, and the results of this analysis are summarized in the heat map shown in FIG.41C, with the OCT4, SOX2, NANOG, and ZFP42 genes being downregulated during differentiation, the ZFPM1, NFE2, RUNX1, MEIS1, and GATA1 genes being upregulated during differentiation, and the PBX1 and MYC genes remaining at a stable level. Petition 870260066603, dated 06 / 07 / 2026, page 37 / 211 23 / 97 substantially consistent.

[0060] FIGS. 42A, 42B, and 42C provide size distributions of PBG1-derived megakaryocytes. FIG. 42A represents a representative example of p1-tubulin staining of PBG1-derived megakaryocytes, which were used to collect size measurements of PBG1-MKs and compare them with MKs from other sources. FIG. 42B represents the size distribution of PBG1-derived megakaryocytes, including the median, quartiles, and range. FIG. 42C compares the size distribution data of PBG1-derived MKs with megakaryocytes from various bone marrow sources.

[0061] FIGS. 43A and 43B provide ploidy measurements in PBG1-derived megakaryocytes. FIG. 43A represents a representative example of DNA ploidy measurements performed on PBG1-derived megakaryocytes. FIG. 43B compares DNA ploidy measurements of PBG1-MKs with MKs from other sources.

[0062] FIG. 44 provides a comparison of the presence or absence and concentration range of various factors in the hiPSC-MK lysate of megakaryocytes derived by a method of the present disclosure and certain controls.

[0063] FIGS. 45A, 45B and 45C represent the production of hiPSC platelets. FIG. 45A represents the flow cytometric analysis of anucleated cells and Petition 870260066603, dated 06 / 07 / 2026, page 38 / 211 24 / 97 nucleated (top left). The nucleated cells contained a large number of CD41+CD42+ megakaryocytes (top right). Anucleated cells positive for CD41+, CD42+, and Calcein AM were evaluated by flow cytometry, and platelets were delineated by size (1-5 microns). FIG. 45B shows examples of platelets harvested from megakaryocyte culture evaluated by electron microscopy. FIG. 45C is a graph representing the cumulative yield of CD41+CD42+Calcein AM+ platelet-sized particles per well during Phase III of the targeted differentiation protocol described herein.

[0064] Although the previously identified designs establish currently disclosed embodiments, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments may be devised by those skilled in the art that fall within the scope and principles of the embodiments disclosed in this document. DETAILED DESCRIPTION

[0065] This disclosure is directed to compositions and methods for the production of megakaryocytic progenitors (preMKs) and megakaryocytes (MKs) from stem cells, such as pluripotent stem cells, by Petition 870260066603, dated 06 / 07 / 2026, page 39 / 211 25 / 97 example, clinical-grade pluripotent stem cells induced by humans. The methods allow the continuous production of preMKs from hemogenic endothelial cells for extended periods (up to 8 days or more), which can be subsequently differentiated into mature MKs. The preMKs and MKs derived by the current methods can be distinguished by one or more of the following: size range, ploidy profile, biomarker expression, gene expression, granule and growth factor composition, cytokine and chemokine composition, or combinations thereof. This disclosure also provides compositions comprising preMKs and MKs and their lysates, as well as methods for using preMKs, MKs, their lysates, and compositions thereof. Definitions

[0066] Unless otherwise defined, all technical and scientific terms used in this document have the meaning commonly understood by a person skilled in the art to which this disclosure pertains. The following references provide those skilled in the art with a general definition of many of the terms used in this disclosure: Singleton et 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 Petition 870260066603, dated 06 / 07 / 2026, p. 40 / 211 26 / 97 (1991). As used in this document, the following terms have the meanings assigned to them below, unless otherwise specified.

[0067] Agent means any small molecule chemical compound, antibody, nucleic acid molecule or polypeptide, or fragments thereof.

[0068] The term antibody, as used herein, refers to an immunoglobulin molecule that binds specifically to an antigen. The term antibody fragment refers to a portion of an intact antibody and refers to the antigenic determinant variable regions of an intact antibody.

[0069] By change or alteration is meant an increase or decrease. A change can be 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30% or 40%, 50%, 60% or even 70%, 75%, 80%, 90% or 100%.

[0070] Biological sample means any tissue, cell, fluid or other material derived from an organism.

[0071] A capture reagent is understood to be a reagent that binds specifically to a nucleic acid or polypeptide molecule in order to select or isolate the nucleic acid or polypeptide molecule.

[0072] Cellular composition means any composition comprising one or more isolated cells.

[0073] Cell survival is understood to mean Petition 870260066603, dated 06 / 07 / 2026, page 41 / 211 27 / 97 cell viability.

[0074] As used in this document, “clinical grade” refers to a cell or cell line derived or obtained using current Good Manufacturing Practices (GMP), which permits its clinical use in humans. GMP is a quality assurance system used in the pharmaceutical industry to ensure that the final product meets predefined specifications. GMP covers the manufacture and testing of the final product. It requires traceability of raw materials and also that production follows validated standard operating procedures (SOPs).

[0075] By “detectable levels” it is understood that the quantity of an analyte is sufficient for detection using methods routinely used to perform such analysis.

[0076] “Detect” refers to identifying the presence, absence, or quantity of the object to be detected.

[0077] By “detectable label” is meant a composition which, when attached to a molecule of interest, renders the latter detectable by means of spectroscopy, photochemistry, biochemistry, immunochemistry or chemistry. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin or haptens. Petition 870260066603, dated 06 / 07 / 2026, page 42 / 211 28 / 97

[0078] Disease is understood to be any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include any illness or injury that results in a reduction in the number of cells or biological function, including ischemic injury such as stroke, myocardial infarction, or any other ischemic event that causes tissue damage, peripheral vascular disease, wounds, burns, fractures, contusions, arthritis, and inflammatory diseases.

[0079] Effective quantity means the amount of an agent needed to produce the intended effect.

[0080] A fragment is understood to be a portion of a polypeptide or nucleic acid molecule. This 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. A fragment may 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.

[0081] The terms isolated, purified, or biologically pure refer to material that is free to varying degrees from components that normally accompany it as found in its native state. Isolate indicates a degree of separation from the original source or surroundings. Purify indicates a degree of separation that is greater than isolation. Petition 870260066603, dated 06 / 07 / 2026, p. 43 / 211 29 / 97 A purified or biologically pure protein is sufficiently free of other materials so that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term purified may indicate that a nucleic acid or protein essentially produces a band on an electrophoretic gel.For a protein that can undergo modifications, such as phosphorylation or glycosylation, different modifications can give rise to different isolated proteins, which can be purified separately.

[0082] By isolated polynucleotide is meant a nucleic acid (e.g., DNA) that is free of the genes that, in the naturally occurring genome of the organism from which the nucleic acid molecule of disclosure is derived, flank the gene. The term therefore includes, for example, recombinant DNA that is incorporated into a vector; in a Petition 870260066603, dated 06 / 07 / 2026, page 44 / 211 30 / 97 plasmid or self-replicating virus; or in the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (e.g., a cDNA or a genomic fragment or cDNA produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0083] An “isolated polypeptide” means a dissemination polypeptide that has been separated from its naturally occurring accompanying components. Typically, the polypeptide is isolated when it is at least 60% by weight free of proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of a dissemination polypeptide. An isolated dissemination polypeptide 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. Petition 870260066603, dated 06 / 07 / 2026, page 45 / 211 31 / 97

[0084] The term “hemogenic endothelial cell,” as used in this document, refers to cells capable of differentiating to give rise to hematopoietic cell types or endothelial cell types and which may optionally be derived from pluripotent stem cells. Hemogenic endothelial cells are normally adherent to extracellular matrix protein and / or other hemogenic endothelial cells and may be characterized, in some embodiments, by the expression of CD31 and CD34 markers.

[0085] By “marker” is meant any protein or other epitope having an altered level of expression or activity that is associated with a characteristic or condition.

[0086] The term “megakaryocyte” (MK), as used in this document, refers to a large (e.g., diameter ^10 pm), polyploid hematopoietic cell with the propensity to generate proplatelets and / or platelets. A morphological characteristic of mature MKs is the development of a large multilobed nucleus. Mature MKs may stop proliferating, but continue to increase their DNA content through endomitosis, with a parallel increase in cell size.

[0087] The term “megakaryocytic progenitor” (preMK), as used in this document, refers to a mononuclear hematopoietic cell that is committed to the lineage. Petition 870260066603, dated 06 / 07 / 2026, page 46 / 211 32 / 97 megakaryocytic and is a precursor to mature megakaryocytes. Megakaryocytic progenitors are normally found in (but 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 were derived from pluripotent stem cells.

[0088] The term microparticle refers to a very small (<1 micron) phospholipid vesicle released from a megakaryocyte or other cell. Microparticles may contain genetic material, such as RNA, and may express extracellular markers of their parent cells. Microparticles derived from megakaryocytes and platelets may play a role in several pathways, including hemostasis and inflammation.

[0089] The term platelet (PLT) refers to a cell with a diameter of 1-3 microns that lacks a nucleus but contains RNA. Platelets can express CD41, CD42b, and CD61 on their cell surface. Internally, they contain alpha 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 a pathway for the transport of extracellular material into the cell and the release of material from the granules into the extracellular environment. They function primarily in the regulation of Petition 870260066603, dated 06 / 07 / 2026, page 47 / 211 33 / 97 hemostasis, participating in blood clotting, but they have also been shown to play a role in inflammation.

[0090] The term preplatelet refers to a cell with a diameter of 3-10 microns without a nucleus, but with RNA. Preplatelets are otherwise morphologically and ultrastructurally similar to platelets and constitute an intermediate cellular stage produced by megakaryocytes that separate by cytoskeletal rearrangement to form individual platelets.

[0091] The term proplatelet refers to cytosolic extensions of megakaryocytes or simply released from megakaryocytes. Proplatelets separate through cytoskeletal rearrangement to form preplatelets and individual platelets.

[0092] The term pluripotent stem cell includes embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells and other stem cells having the capacity to form cells of all three germ layers of the body, regardless of the method by which the pluripotent stem cells are derived. Pluripotent stem cells are functionally defined as stem cells that may have one or more of the following characteristics: (a) being able to induce teratomas when transplanted into immunodeficient mice (SCID); (b) being able to Petition 870260066603, dated 06 / 07 / 2026, page 48 / 211 34 / 97 differentiate into cell types from all three germ layers (e.g., they can differentiate into ectodermal, mesodermal, and endodermal cell types); or (c) express one or more embryonic stem cell markers (e.g., express Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, SSEA-5 surface antigen, Nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.).

[0093] The term “induced pluripotent stem cells” (iPS cells or iPSCs) refers to a type of pluripotent stem cell generated by reprogramming a somatic cell through the expression of a combination of reprogramming factors. iPSCs can be generated using fetal, postnatal, newborn, juvenile, or adult somatic cells. Factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct 4 (sometimes referred to as Oct 3 / 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 Oct 4, Sox2, Nanog, and Lin28. In certain forms of embodiment, at least two, three, or four reprogramming factors are expressed in a somatic cell to reprogram the somatic cell.

[0094] As used in this document, the terms Petition 870260066603, dated 06 / 07 / 2026, page 49 / 211 35 / 97 prevent, preventing, prevention, “prophylactic treatment and the like” refer to reducing the likelihood of developing a disorder or condition in an individual who does not have, but is at risk of having, or is susceptible to developing, a disorder or condition.

[0095] By reduction is meant a negative change of at least 10%, 25%, 50%, 75% or 100%.

[0096] By reduction of cell death is meant the reduction of the propensity or probability of a cell dying. Cell death can be apoptotic, necrotic, or by any other means.

[0097] By reduced level it is understood that the amount of an analyte in a sample is less than the amount of the analyte in a corresponding control sample.

[0098] By reference is meant a standard or a control condition.

[0099] By specifically binding is meant a compound or antibody that recognizes and binds to a disclosure polypeptide, but does not substantially recognize and bind to other molecules in a sample, for example, a biological sample, which naturally includes a disclosure polypeptide.

[00100] The term individual or patient refers to an animal that is the subject of treatment, observation, or experimentation. As an example, an individual includes, Petition 870260066603, dated 06 / 07 / 2026, p. 50 / 211 36 / 97 but not limited to, a mammal, including, but not limited to, a human or non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine or feline.

[00101] As used in this document, the terms treat, treating, treatment and the like refer to reducing or improving a disorder or symptoms associated with it. It will be appreciated that, while not prohibited, the treatment of a disorder or condition does not require that the disorder, condition or associated symptoms be completely eliminated.

[00102] By, includes, comprising, containing and having and the like may have the meaning ascribed to them in US patent law and may mean includes, including and the like; Consisting essentially of or essentially consists of in the same way has the meaning ascribed to it in US patent law and the term is open-ended, allowing for the presence of more than what is indicated, provided that basic or new features that are indicated are not altered by the presence of more than what is indicated, but excludes prior art embodiments.

[00103] Unless specifically stated or obvious from the context, as used in this document, the term "or" is understood to be inclusive. Unless Petition 870260066603, dated 06 / 07 / 2026, page 51 / 211 37 / 97 as specifically stated or obvious from the context, as used in this document, the terms a, an and the are understood in the singular or plural.

[00104] Unless specifically stated or obvious from the context, as used in this document, the term "about" is understood to mean within a normal tolerance range in the technique, for example, within 2 standard deviations of the mean. "Over" may be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated Value. Unless otherwise clear from the context, all numerical values ​​given in this document are modified by the term "about".

[00105] The recitation of a list of chemical groups in any definition of a variable in this document includes definitions of that variable as any single group or combination of groups listed. The recitation of an embodiment for a variable or aspect in this document includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[00106] Any compositions or methods provided in this document may be combined with one or more of any of the other compositions and methods provided in this document. Megakaryocytes Petition 870260066603, dated 06 / 07 / 2026, page 52 / 211 38 / 97

[00107] In the human body, megakaryocytes are derived from CD34+ hematopoietic stem cells that reside primarily in the bone marrow, but are also found in the yolk sac, fetal liver, and spleen during early development. During MK differentiation, MK precursors undergo a period of endomitosis, in which MKs become polyploid through multiple cycles of DNA replication without cell division, producing a polylobed nucleus with up to 128n copies of DNA. As MKs increase in size, they also refine their transcriptional and proteomic profiles, acquire a series of highly specialized granules, including α-granules and dense granules, and develop a highly invaginated membrane system, which are hallmarks of MK maturation and development.

[00108] To produce platelets, MKs migrate adjacent to blood vessels in the bone marrow, through which they extend long structures called proplatelets into the circulation. Proplatelets function as assembly lines for platelet production and sequentially release multiple anucleated platelets from their ends.

[00109] Although platelets are primarily responsible for clot formation at sites of active bleeding, it is becoming increasingly apparent Petition 870260066603, dated 06 / 07 / 2026, page 53 / 211 39 / 97 that they also play significant roles in wound healing, angiogenesis, and innate immunity.In the stem cell-based therapeutic landscape, platelets are an ideal initial candidate because: 1) they are in high clinical demand due to their short registration shelf life; 2) they are anucleated and can be safely irradiated to kill any contaminating nucleated cells, thus reducing the risk of teratoma development; 3) they do not require HLA or blood type matching for most (>90%) platelet transfusions, which will facilitate large-scale manufacturing of allogeneic human pluripotent stem cells (hPSCs) for off-the-shelf therapy; 4) they have a short half-life and are well-characterized, which will simplify designed clinical trials; 5) they are easily transplanted; and 6) they would benefit enormously from sterile manufacturing, as current donor-based platelet transfusions are inherently susceptible to bacterial and viral contamination.

[00110] Current platelet demand exceeds supply by ~20%, and unmet demand is expected to more than double by 2022 due to population growth and aging requiring more platelet-based procedures, new medications that reduce platelet counts, and the expansion of existing uses of platelet transfusions to improve healing time. In the United States, Petition 870260066603, dated 06 / 07 / 2026, page 54 / 211 40 / 97 the blood market effectively operates as an oligopoly, with the American Red Cross and Blood Centers of America each controlling slightly less than half of the market and HemeXcel as the third largest blood provider.

[00111] There are several 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 dispensing, and in the cosmetics industry for skin rejuvenation. Because megakaryocytes produced by the methods described in this document contain many growth factors, they can be a source of therapeutic compositions and / or used for many therapeutic purposes. This disclosure addresses these needs by establishing a scalable commercial platform compliant with current Good Manufacturing Practices (cGMP) for making human iPSC-derived megakaryocytes and products thereof.

[00112] Outside the body, MKs can reasonably be derived from several major stem cell sources, for example, pluripotent stem cells, hematopoietic stem cells, or other types of stem cells.

[00113] In some embodiments, MKs may be derived from pluripotent stem cells, including, Petition 870260066603, dated 06 / 07 / 2026, page 55 / 211 41 / 97, 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-stage 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 and maintained in culture indefinitely and engineered to produce MKs.

[00114] In some embodiments, MKs may be derived from hematopoietic stem cells, including, but not limited to, CD34+ umbilical cord blood stem cells (UCB cells) (e.g., CD34+ umbilical cord blood stem cells), CD34+ mobilized peripheral blood cells (MPB cells) (e.g., CD34+ human mobilized peripheral blood), or CD34+ bone marrow cells. UCB cells are multipotent stem cells derived from blood remaining in the placenta and attached umbilical cord after delivery. MPB cells are multipotent stem cells derived from volunteers whose stem cells are mobilized into the bloodstream by administration of G-CSF or a similar agent.

[00115] In some embodiments, MKs can Petition 870260066603, dated 06 / 07 / 2026, page 56 / 211 42 / 97 be derived from other types of stem cells, including, but not limited to, mesenchymal stem cells (MSCs) (such as adipose tissue-derived mesenchymal stem cells (AdMSCs)) or mesenchymal stem cells from other sources.

[00116] AdMSCs are derived from white adipose tissue, which is derived from the mesoderm during embryonic development and is present in all mammalian species, located throughout the body. Due to their wide availability and their ability to differentiate into other tissue types, ASCs of bone, cartilage, muscle, and adipose tissue, including mesoderm, can serve a wide variety of applications.

[00117] In the present disclosure, stem cell cultures are maintained independently of embryonic fibroblast feeder cells and / or animal serum which may be potentially contaminated with xenogeneic pathogens and may increase the risk of an immunogenic reaction in humans. Therefore, serum- and feeder cell-free alternatives are used in the present methods to avoid the introduction of animal products into preMKs and MKs derived according to the present methods to ensure safe and animal product-free conditions and products. Production Methods Petition 870260066603, dated 06 / 07 / 2026, page 57 / 211 43 / 97

[00118] FIG. 1 shows a general scheme for scalable differentiation of megakaryocytic progenitors (preMKs), megakaryocytes (MKs), and platelets (PLTs) from one or more pluripotent stem cells. However, it should be noted that, although the present processes are described in connection with pluripotent stem cells, in various embodiments, pluripotent stem cells may be replaced or supplemented with other types of stem cells. Phase 0: Expansion of human induced pluripotent stem cells and preparation for differentiation. Matrix-dependent expansion crops

[00119] For matrix-dependent expansion cultures, clinical-grade pluripotent stem cells (PSCs) can be expanded as colonies by culturing without feeder cells in a support matrix in a pluripotent stem cell culture medium. The support matrix can be a two-dimensional surface or a three-dimensional structure. In some embodiments, clinical-grade human induced pluripotent stem cells may be human induced pluripotent stem cells (iPSCs), such as PBG1, PBG2, or PBG3, but other types of pluripotent stem cells, such as embryonic stem cells, or other stem cells may be used.

[00120] In some embodiments, the matrix of Petition 870260066603, dated 06 / 07 / 2026, page 58 / 211 44 / 97 support may be, by way of non-limiting example, recombinant vitronectin, recombinant laminin, Matrigel, or any combination thereof. 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 medium capable of supporting the maintenance and growth of pluripotent cells known in the art. In some embodiments, cells may be cultured to achieve confluence. In some embodiments, cells may be cultured to achieve 30% to 90% confluence. In some embodiments, cells are cultured to achieve up to 60%, up to 65%, up to 70%, up to 75% confluence. For example, cells are cultured to achieve approximately 70% confluence. When a predetermined maximum percentage of confluence is reached, the cells are harvested.In some embodiments, cells can be harvested as clumps by dissociation using 0.1 mM to 5 mM EDTA or a chelating agent or similar reagent. For example, cells can be harvested using approximately 0.5 mM EDTA. In some embodiments, cells can be harvested as single cells, such as, for example, by dissociation with proteolytic enzymes, collagenolytic enzymes, or combinations thereof. For example, as... Petition 870260066603, dated 06 / 07 / 2026, page 59 / 211 45 / 97 cells can be harvested as individual cells by dissociation with, for example, recombinant trypsin, such as TrypLETM or AccutaseTM. For maintenance / expansion of PSCs, the harvested cells can be resuspended in pluripotent stem cell culture medium. Matrix-independent 3D expansion cultures

[00121] For matrix-independent 3D expansion cultures, clinical-grade PSCs can be expanded as self-aggregating spheroids. In some embodiments, this can be achieved by seeding individual cells at a density of about 0.1 million to about 1.5 million per ml. For example, in some embodiments, individual cells can be seeded at 0.5 million per ml.

[00122] Cells can be subjected to continuous movement by slow agitation or gentle agitation under low-adherence or non-adherence conditions in a pluripotent stem cell culture medium. In some embodiments, feeder-free and serum-free media may be used. 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 similar media capable of supporting the maintenance and growth of pluripotent cells known in the art. In some embodiments of Petition 870260066603, dated 06 / 07 / 2026, page 60 / 211 In embodiment 46 / 97, PSC spheroids are cultured until they reach a total cell density of about 3 to about 10 million cells / ml and / or an average spheroid size of about 150 to about 350 μm, for approximately 5-7 days. In some embodiments, PSC spheroids are cultured until they reach a total cell density of 5 million cells / ml. In some embodiments, PSC spheroids are cultured until the cells reach an average spheroid size of about 250 μm. The culture step may last 4, 5, 6, 7, or 8 days. When applicable, PSCs can be harvested as individual cells by dissociation with proteolytic enzymes, collagenolytic enzymes, or combinations thereof. For example, cells can be harvested as single cells by dissociation with, but not limited to, trypsin, recombinant trypsin such as TrypLET™, Accutase™, or a similar reagent known in the art.In some embodiments, individual cells are used to initiate another 3D expansion culture and / or targeted differentiation culture. Preparation for Differentiation

[00123] In some embodiments, to prepare for differentiation, PSC aggregates can be generated by partial dissociation of PSC colonies from matrix-dependent 2D cultures, by partial dissociation Petition 870260066603, dated 06 / 07 / 2026, page 61 / 211 47 / 97 of PSC spheroids from matrix-independent 3D cultures or by self-aggregation of individual PSCs generated by any method known in the art. In some embodiments, before the onset of differentiation, these aggregates can be resuspended and cultured in a pluripotent stem cell culture medium, for example, but not limited to, Essential 8 medium (ThermoFisher), StemFlex medium (ThermoFisher) or NutriStem medium (Biological Industries). In some embodiments, the medium may include a ROCK inhibitor, such as, for example, but not limited to, Y27632, H1152 or a combination thereof. In some embodiments, the cells can be cultured for between 0 and 72 hours at 37 °C, 5% CO2, 20% O2 before the onset of differentiation.

[00124] For matrix-dependent cultures, aggregates can be affixed to a surface. In some embodiments, the affixation step can proceed for about 24 hours, although any time between 1 hour and 24 hours or more 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 collagen IV can be used to coat the surface. In some embodiments, the matrix-coated surface can be 2D (e.g., the bottom of a dish or flask). Petition 870260066603, dated 06 / 07 / 2026, page 62 / 211 48 / 97 plastic). In some embodiments, the matrix-coated surface may be 3D (e.g., smooth or textured spherical microcarriers, microcarriers such as Rauchig rings). Cells on the 3D matrix-coated surfaces can then be cultured with or without continuous movement. For example, cells can be cultured under static ultra-low adhesion conditions in spin flasks, screw flasks, rotating flasks, shake tank bioreactors, vertical wheel bioreactors, packed bed bioreactors, or fluidized bed systems.

[00125] For matrix-independent cultures, aggregates can be subjected to continuous movement by slow agitation or gentle agitation in a low-stick vessel. Cells can transition to Phase I of differentiation after between 0 and 72 hours, for example, after about 24 hours. Phase I. Generation of Hemogenic Endothelial Cells

[00126] 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 (ACF) medium comprising StemSpan™-ACF. Petition 870260066603, dated 06 / 07 / 2026, page 63 / 211 49 / 97 (STEMCELL Technologies, Cat. No. 09855) as 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 between 1 and 200 ng / ml of one or more of each of BMP4 (e.g., at 50 ng / ml), bFGF (e.g., at 50 ng / ml), and VEGF (e.g., at 50 ng / ml). Cells can be incubated for between 2 and 6 days under low oxygen conditions (e.g., 37°C, 5% CO2, 5% O2), followed by between 2 and 6 days under normoxic conditions (37°C, 5% CO2, 20% O2). In some embodiments, WNT modulators, such as WNT agonists or antagonists, may be added during the initial differentiation period. In some embodiments, GSK3 inhibitors may be added during the initial differentiation period.In some embodiments, GSK3 inhibitors or WNT agonists, such as, for example, CHIR9998014, CHIR99021, or a combination thereof, may be added for the initial differentiation period, such as between 1 and 2 days. In some embodiments, WNT modulators may replace one or more of the growth factors for at least a portion of Phase I. For example, in some embodiments, while the WNT modulators are... Petition 870260066603, dated 06 / 07 / 2026, page 64 / 211 In some embodiments, VEGF and bFGF may be dispensable during the first 48 hours while WNT modulators are present. In some embodiments, complete daily medium changes may be performed throughout Phase I by removing spent medium and replacing it with freshly prepared Phase I medium. In some embodiments, partial medium changes may be performed, with 10-95% of the spent medium removed and replaced with equivalent volumes of Phase I medium. In some embodiments, additional volumes of freshly prepared medium may be added with the net effect of increasing the total culture volume. In some embodiments, specific medium components are added to the culture instead of replacing or adding Phase I medium.

[00127] In 2D matrix-dependent cultures, on day 2, the morphology of the colonies changes to clusters of scattered elongated cells (FIG. 15). On days 5-6, a confluent adherent layer of hemogenic endothelial cells is observed, with some three-dimensional structure within the adherent cell layer (FIG. 15). In matrix-independent 3D cultures, the spheroids become larger, darker, and less uniform as Phase I progresses (FIG. 33A). Approximately 6 days after the start of the Petition 870260066603, dated 06 / 07 / 2026, page 65 / 211 51 / 97 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 structure within the adherent cell layer (FIG. 4). To confirm successful Phase I differentiation, a portion of the cells can be harvested as individual 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 a similar reagent known in the art, followed by flow cytometry analysis for the hemogenic endothelium-specific markers CD31 and CD34. In some embodiments, hemogenic endothelial cells may also express CD309 and CD144 or CD309, CD144, CD140a, and CD235a.In some embodiments, Phase I can be carried out in a stirred tank bioreactor to form self-aggregating spheroids. Phase II. Generation of Separate Megakaryocytic Progenitors (preMKs) from Hemogeneous Endothelial Cells

[00128] In some embodiments, the initiation of megakaryocytic progenitor differentiation (Phase II) can be performed after 4 and 8 days of Phase I. Briefly, the middle of Phase I is removed and replaced with a volume Petition 870260066603, dated 06 / 07 / 2026, page 66 / 211 52 / 97 equivalent of Phase II medium, such as, for example, STEMdiff™ APEL™2 basal medium (STEMCELL Technologies, Cat. No. 05275). Such Phase II medium may be supplemented with 1 and 200 ng / ml of each or more of Stem Cell Factor (SCF) (e.g., at 25 ng / ml), Thrombopoietin (TPO) (e.g., at 25 ng / ml), Fms-related tyrosine kinase ligand 3 (Flt3-L) (e.g., at 25 ng / ml), Interleukin-3 (IL-3) (e.g., at 10 ng / ml), Interleukin-6 (IL-6) (e.g., at 10 ng / ml) and heparin (e.g., at 5 units / ml). In some embodiments, the Phase II medium may be further supplemented with UM171, UM729, SR-1, SU6656, or any combination thereof.

[00129] The cells are then incubated for at least 7 and up to 12 or more days at 37 °C, 5% CO2, 20% O2. For example, cells may be incubated for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 days. In some embodiments, daily partial medium changes may be performed, with 10-95% of the spent medium removed and replaced with equivalent volumes of freshly prepared Phase II medium. In some embodiments, additional volumes of freshly prepared medium may be added with the net effect of increasing the total culture volume. In some embodiments, specific medium components may be added to the culture instead of replacing or adding freshly prepared Phase I medium. Petition 870260066603, dated 06 / 07 / 2026, page 67 / 211 53 / 97

[00130] Within 2-3 days after the start of Phase II, small, round, refractile cells appear within the adherent hemogenic endothelial cells and are eventually released into the supernatant (FIG. 17A). These released cells may contain preMKs, as defined by cell surface expression of CD43 and CD41 and without CD14 expression. In 2D cultures, the floating and weakly attached Phase II cells that appear on top of the adherent cell layer can be harvested daily by gentle rinsing and collecting the medium in conical tubes. A half-measurement change can be initiated by adding half the original volume of freshly prepared Phase II medium on top of the rinsed adherent cell layer. An aliquot of the cells collected in the medium can be removed for viable cell enumeration and biomarker analysis by flow cytometry. The remaining cells can then be centrifuged at 200xg for 5 minutes.After centrifugation, a medium change can be completed by adding back half the original volume of supernatant to the layer of adherent cells. In matrix-independent 3D cultures, the released cells can be harvested by pausing agitation to allow the spheroids to settle to the bottom of the vessel, then collecting up to 90% of the medium, along with the suspended cells, into tubes (e.g., conical tubes) for centrifugation. Half the original volume of freshly arrived medium. Petition 870260066603, dated 06 / 07 / 2026, page 68 / 211 Prepared 54 / 97 medium can then be added to the vessel and topped up with conditioned medium from the supernatant of the centrifuged cells. In some embodiments, additional volumes of freshly prepared medium can be added with the net effect of increasing the total culture volume. In some embodiments, specific medium components can be added to the culture instead of replacing or adding freshly prepared Phase II medium. The remaining supernatant is discarded, and the cell pellet containing preMK can be stored at -180 °C in Criostor 10 cryopreservation medium or directly transitioned to Phase III. The megakaryocytic progenitors collected during Phase II are small, round, refractile cells that express CD43 and CD41 and do not express CD14. Phase III. Generation of mature megakaryocytes (MK) from megakaryocytic progenitors.

[00131] In some embodiments, the differentiation of mature megakaryocytes can be initiated using PSC-derived preMKs, generated as described above. Freshly prepared or thawed megakaryocytic progenitors can be seeded onto a non-adherent surface in Phase III medium, comprising, for example, StemSpan™-ACF. Non-adherent surfaces refer to surfaces such that most cells are not intended to adhere. Petition 870260066603, dated 06 / 07 / 2026, page 69 / 211 55 / 97 cells adhere or stick to such surfaces, but instead remain primarily in suspension. For example, this 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, the Phase III medium may be supplemented with, between 0 and 200 ng / ml each, of one or more of TPO (e.g., at 25 ng / ml), SCF (e.g., at 25 ng / ml), IL-6 (e.g., at 10 ng / ml), IL-9 (e.g., at 10 ng / ml), and Heparin (e.g., at 5 units / ml). In some embodiments, the Phase III medium may also be supplemented with UM171, UM729, SR1, SU6656, or any combination thereof.

[00132] The cells can then be incubated 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 for up to 5 days. In some embodiments, partial daily medium changes are performed, with 10-95% of the spent medium removed and replaced with equivalent volumes of freshly prepared Phase III medium. In some embodiments, the non-adherent surface is a 6-well ultralow adhesion plate. In some embodiments, the non-adherent surface is a gas-permeable membrane (such as GRex®). In some embodiments, the non-adherent surface is a cell culture bag or vessel with Petition 870260066603, dated 06 / 07 / 2026, page 70 / 211 56 / 97 gentle stirring.

[00133] In some embodiments, during Phase III, megakaryocytic progenitors can differentiate into mature MKs within several days. In some embodiments, cells that are initially uniformly small, round, and refractile (FIG. 21) begin to increase in size and ploidy on days 2-4 (FIG. 20). Simultaneously, proplatelet-producing MKs can be readily observed (FIG. 20). By days 3-4 of Phase III, the proportion of CD61+ cells (megakaryocytic lineage) co-expressing the mature MK markers CD42a and CD42b increases dramatically and can reach levels as high as 80-90%, depending on the initial hiSPC cell line (FIG. 21). On days 4-5 of Phase III, platelets are released by mature MKs into the culture medium. These platelets can be collected, quantified, and evaluated by flow cytometry and electron microscopy, confirming their identity as genuine platelets (FIG. 45). 3D Systems Packed bed bioreactor

[00134] In some embodiments, a scalable 3D packed bed bioreactor can be used for the production of one or more preMKs, megakaryocytes, platelets, or megakaryocytes and platelets. In some embodiments, the packed bed bioreactor can be used Petition 870260066603, dated 06 / 07 / 2026, page 71 / 211 57 / 97 for Phase I and Phase II culture. For example, the fixed-bed reactor can be used for the differentiation of PSCs into hemogenic endothelial cells, followed by the production of preMKs. Fixed-bed reactor carriers can be micro or macro-sized and can be formed from biocompatible plastics, metals, glass, or natural materials such as alginate. In some embodiments, the carriers are formed from PTFE in the form of Raschig rings, for example, 1 mm Raschig rings. In some embodiments, the carriers can be coated with a matrix as described above. In some embodiments, the carriers can be coated with Laminin, such as a recombinant human protein Laminin 521. In some embodiments, pluripotent cells can be seeded as clusters in the carriers. In some embodiments, the medium can be removed and replaced with Phase I medium, with daily medium changes.In some embodiments, during Phase I, pluripotent cells may exhibit growth areas within the carriers in the fixed-bed reactor. In some embodiments, the initial differentiation of pluripotent cells to hemogenic endothelium (i.e., Phase I of directed differentiation), as well as the further differentiation and release of preMKs (i.e., Phase II of directed differentiation) may occur in the same vessel. For example, a. Petition 870260066603, dated 06 / 07 / 2026, page 72 / 211 A 58 / 97 fixed-bed bioreactor may comprise macrocarriers coated with Laminin-521 seeded with pluripotent cells, for example PBG-1 iPSCs. The packed bed can then be exposed to a continuous flow of medium to allow Phase I differentiation of the hemogenic endothelium. After percolating through the packed bed, the medium can be circulated through a conditioning chamber, where freshly prepared medium components can be added and oxygen / CO2 concentrations can be adjusted by spraying or other means before the medium can be recirculated to the cells.

[00135] At the conclusion of Phase I, the medium can be changed to allow for Phase II differentiation and the production and release of preMKs. Suitablely sized and shaped carriers, such as 1 mm Raschig rings, can allow for sufficient medium flow and channel width to permit the released cells to percolate through the packed bed and exit the reactor for collection and cryostorage. In some embodiments, this design can decrease the shear forces experienced by the cells, can allow for efficient medium use due to its perfusion-based design, and can allow for continuous collection of preMKs as they are released. Self-assembling spheroids in a stirred tank bioreactor Petition 870260066603, dated 06 / 07 / 2026, page 73 / 211 59 / 97

[00136] In some embodiments, certain steps of the process can be performed using a scalable 3D solution, which may involve performing differentiations using self-aggregating spheroids suspended in agitated or stirred vessels (FIG. 32). In some embodiments, these vessels may include low-adherent or non-adherent surfaces, i.e., surfaces coated with hydrophilic or neutrally charged coatings to inhibit the immobilization of specific and non-specific cells on the surface, forcing the cells into a suspended state. Pluripotent cells can be dissociated into single cells and resuspended in pluripotency maintenance medium. In some embodiments, the maintenance medium may be supplemented with a ROCK inhibitor, such as H1152 or another ROCK inhibitor.Pluripotent cells can then be incubated in a low-adherent or non-adherent vessel and subjected to agitation under standard culture conditions (e.g., 37 °C, 5% CO2, 20% O2). In some embodiments to provide agitation, the incubation vessel can be placed on an orbital shaker, or a shaker flask or rotating flask with constant agitation, or a controlled-stir tank bioreactor can be used. Within 24 hours, the pluripotent cells can self-aggregate to form spheroids of approximately 50–150 µm. Petition 870260066603, dated 06 / 07 / 2026, page 74 / 211 60 / 97 diameter. When agitation is stopped, the spheroids may settle to the bottom of the vessel.

[00137] The media can then be exchanged with Phase I differentiation media to promote differentiation towards hemogenic endothelium, and agitation can be resumed, with incubation under hypoxic conditions (e.g., 37°C, 5% CO2, 5% O2). Media exchanges can be performed regularly (e.g., daily), during which the spheroids can grow and develop characteristic structure and shapes. For example, as shown in FIG. 33A, the 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 FIG. 33A, on day 6 the spheroids are larger, darker, and have an irregular surface.

[00138] To transition to Phase II, agitation can be stopped and the spheroids can settle to the bottom of the vessel. The media can then be exchanged for Phase II differentiation media to promote differentiation and release of cells in suspension. Subsequently, on a regular basis (e.g., daily), cells in suspension can be collected and a partial medium exchange can be performed. The medium can be collected and centrifuged. Approximately half the working volume of the freshly prepared Phase II differentiation medium Petition 870260066603, dated 06 / 07 / 2026, page 75 / 211 61 / 97 can be added to the spheroids, along with a sufficient volume of conditioned medium (i.e., post-centrifugation supernatant) to restore the original working volume. The cell beads can be cryopreserved or transferred to Phase III for maturation into mature MKs.

[00139] After transitioning to static Phase III cultures, preMKs from 3D self-aggregating spheroid cultures can generate MK purities similar to those of preMKs from 2D culture systems. Furthermore, Phase III differentiation cultures generated from 3D self-aggregating spheroid cultures can contain cells that have dramatically increased in size and are capable of generating proplatelets, consistent with their identity as genuine megakaryocytes. Transition to a scalable system for Phase III

[00140] In some embodiments, as noted above, freshly prepared or thawed megakaryocytic preMKs can be seeded onto a low-adherent or non-adherent surface in Phase III medium. In some embodiments, this non-adherent surface can be a gas-permeable membrane (such as G-Rex®). In some embodiments, the low-adherent or non-adherent surface is a gently agitated cell culture bag or vessel. In both cases, the preMKs (freshly harvested from Phase II culture or thawed from Petition 870260066603, dated 06 / 07 / 2026, page 76 / 211 62 / 97 cryopreserved loads) are suspended in Phase III medium at a density of 0.5–10 million per ml and introduced into the vessel. For example, preMKs may be at densities of 1–1.5 million per ml, 1–2 million per ml, 1–3 million per ml, 1–4 million per ml, 2–5 million per ml, 2–6 million per ml, 3–7 million per ml, 3–8 million per ml, 5–9 million per ml, or 8–10 million 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, half-measurement exchanges are performed daily, with 10–95% of the spent medium removed and replaced with equivalent volumes of freshly prepared Phase III medium. At the end of Phase III cultures, the resulting cells are increased in size and ploidy, and exhibit host characteristics indicative of mature megakaryocytes (as, for example, shown in FIGS).34 to 42 and described below). Megakaryocytes and their Products

[00141] In some embodiments, the present disclosure provides a megakaryocytic progenitor, a megakaryocyte, preplatelets, proplatelets, or a platelet derived in vitro from a PSC cell or cell line. According to aspects of the present disclosure, the megakaryocytic progenitor, a megakaryocyte, preplatelets, Petition 870260066603, dated 06 / 07 / 2026, page 77 / 211 63 / 97 Proplatelets or a platelet derived from a PSC cell or cell line are produced using the method of U.S. Patent No. 9,763,984 or the bioreactor as disclosed in International Application No. PCT / US2018 / 021354, which are incorporated herein by reference in their entirety.

[00142] In some embodiments, the present disclosure provides an isolated population of cells comprising the megakaryocyte or megakaryocytic progenitor.

[00143] In some embodiments, the present disclosure provides a composition containing a megakaryocyte or megakaryocytic. In some embodiments of the present disclosure, the composition comprising megakaryocytes, megakaryocytic progenitor or products thereof is disclosed.

[00144] According to some embodiments of the present disclosure, the megakaryocyte, megakaryocytic progenitor, or products thereof are homogeneous in shape, size, and / or phenotype. It should be appreciated that the megakaryocyte, megakaryocytic progenitor, or products thereof of the present disclosure may comprise variability in biomarker expression, size, ploidy, number, and purity that is characteristically different from the variability in the corresponding human cells. In some embodiments, this variability may be significantly smaller. In some Petition 870260066603, dated 06 / 07 / 2026, page 78 / 211 64 / 97 embodiments, cell populations can be created to have a desired variability, which can be less or greater than that of naturally occurring cells.

[00145] In some embodiments, megakaryocytic progenitors (preMKs) are characterized by the expression of CD43 and CD41 markers and the lack of CD14 (i.e., CD14-, CD41+, CD43+). Additional expression of CD42b may indicate that the megakaryocytic progenitor is in the final maturation process towards mature megakaryocytes. In certain embodiments, megakaryocytic progenitors generated in differentiation cultures are non-adherent and may float freely in the culture medium.

[00146] In some embodiments, the present megakaryocytes are one or more CD42a+, CD42b+, CD41+, CD61+, GPVI+, and DNA+. In some embodiments, the present megakaryocytes are one or more CD42a+, CD42b+, CD41+, CD61+, and DNA+. In some embodiments, the present megakaryocytes are one or more CD42b+, CD61+, and DNA+. In some embodiments, the present megakaryocytes are one or more CD42a+, CD61+, and DNA+. In some embodiments, the present megakaryocytes are one or more CD42a+, CD41+, and DNA+. In some embodiments, the present megakaryocytes are one or more CD42b+, CD41+, CD61+, and DNA+. In some embodiments, the megakaryocytes present are one or more CD42b+, CD42a+, CD61+, and Petition 870260066603, dated 06 / 07 / 2026, pp. 79 / 211 65 / 97 DNA+. In some embodiments, the megakaryocytes present are one or more CD42b+, CD42a+, CD41+, and DNA+. In some embodiments, the megakaryocyte is CD41+CD61+CD42b+GPVI+. In some embodiments, the megakaryocyte is CD41+CD61+CD42a+GPVI+.

[00147] In some embodiments, the present megakaryocyte is CD61+ and DNA+ and has a diameter of about 10-50 pm. In some embodiments, the megakaryocytes produced by the methods described in this document have an average size between 10 and 20 pm, between 11 and 19 pm, between 12 and 18 pm, between 13 and 17 pm, between 14 and 16 pm, between 14 and 15 pm. In some embodiments, the megakaryocytes produced by the methods described in this document have an average size of 14.5 pm. In some embodiments, the present megakaryocyte has a diameter of about 10-20 pm. In some embodiments, the present megakaryocyte has a diameter of about 10-30 pm. In some embodiments, the present megakaryocyte has a diameter of about 10-40 pm. In some embodiments, the present megakaryocyte has a diameter of about 10-50 µm. In some embodiments, the present megakaryocyte has a diameter of about 20-40 µm.In some embodiments, the present megakaryocyte has a diameter of about 25-40 µm.

[00148] In some embodiments, the gifts Petition 870260066603, dated 06 / 07 / 2026, pages 80 / 211 66 / 97 megakaryocytes produced by the methods described in this document have a ploidy of 2N-16N. In some embodiments, the present megakaryocyte has a ploidy of at least 4N, 8N, or 16N. In some embodiments, the present megakaryocytes have a ploidy of 4N-16N. In some embodiments, the present megakaryocytes produced by the methods described in this document are 16% + / -11.4% CD61+ cells in 72 hours of Phase III culture with DNA greater than 4N.

[00149] In some embodiments, at least 50% of the megakaryocyte population produced by the methods described in this document is CD61+ and DNA+, and has a ploidy of 2N to 16N. For example, megakaryocytes (i.e., Phase III beta-1-tubulin-positive cells) from a representative PBG1 differentiation culture ranged in size from about 9 µm to about 27 µm, with a median of 15 µm. This average size compares similarly with normal megakaryocytes from various bone marrow sources (FIG. 41).

[00150] In some embodiments, the isolated cell population or composition contains at least 50% CD42b+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 55% CD42b+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition Petition 870260066603, dated 06 / 07 / 2026, page 81 / 211 67 / 97 contains at least 65% CD42b+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 60% CD42b+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 70% CD42b+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 75% CD42b+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 80% CD42b+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 85% CD42b+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 90% CD42b+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 95% CD42b+CD61+ DNA+ cells.In some embodiments, the isolated cell population or composition contains at least 98% CD42b+CD61+ DNA+ cells.

[00151] In some embodiments, the isolated cell population or composition contains at least 50% CD42b+CD41+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 55% CD42b+CD41+CD61+ DNA+ cells. In some embodiments, the isolated cell population Petition 870260066603, dated 06 / 07 / 2026, page 82 / 211 68 / 97 or the composition contains at least 65% CD42b+CD41+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 60% CD42b+CD41+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 70% CD42b+CD41+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 75% CD42b+CD41+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 80% CD42b+CD41+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 85% CD42b+CD41+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 90% CD42b+CD41+CD61+ DNA+ cells.In some embodiments, the isolated cell population or composition contains at least 95% CD42b+CD41+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 98% CD42b+CD41+CD61+ DNA+ cells.

[00152] In some embodiments, the isolated cell population or composition contains at least 50% CD42b+CD42a+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 55% CD42b+CD42a+CD61+ DNA+ cells. In Petition 870260066603, dated 06 / 07 / 2026, page 83 / 211 69 / 97 In some embodiments, the isolated cell population or composition contains at least 65% CD42b+CD42a+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 60% CD42b+CD42a+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 70% CD42b+CD42a+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 75% CD42b+CD42a+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 80% CD42b+CD42a+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 85% CD42b+CD42a+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 90% CD42b+CD42a+CD61+ DNA+ cells.In some embodiments, the isolated cell population or composition contains at least 95% CD42b+CD42a+CD61+ DNA+ cells. In some embodiments, the isolated cell population or composition contains at least 98% CD42b+CD41+CD61+ DNA+ cells.

[00153] In some embodiments, the isolated cell population or composition contains at least 50% megakaryocytes with ploidy of 4N or higher. In some embodiments, at least 50% of the megakaryocytes have ploidy Petition 870260066603, dated 06 / 07 / 2026, page 84 / 211 70 / 97 4N-16N. In some embodiments, at least 60% of megakaryocytes have 4N-16N ploidy. In some embodiments, at least 70% of megakaryocytes have 4N-16N ploidy. In some embodiments, at least 80% of megakaryocytes have 4N-16N ploidy. In some embodiments, at least 90% of megakaryocytes have 4N-16N ploidy. In some embodiments, the isolated cell population or composition contains megakaryocytes with an average ploidy of 4N.

[00154] In some embodiments, the isolated cell population or composition contains a proplatelet, preplatelet, or platelet generated from a megakaryocyte of the present disclosure. In some embodiments, the proplatelet, preplatelet, or platelet is a CD42b+CD61+ DNA- cell. In some embodiments, the megakaryocyte is produced in vitro by differentiation of a hiPSC cell or cell line.

[00155] In some embodiments, the megakaryocytes produced by the methods described in this document 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) have low / no fibrinogen, serotonin, and LDL content, and Petition 870260066603, dated 06 / 07 / 2026, page 85 / 211 71 / 97 (d) can capture fibrinogen, serotonin and LDL when incubated with plasma.

[00156] In some embodiments, the megakaryocytes produced by the methods described in this document have a characteristic expression profile of growth factors, cytokines, chemokines and related factors (FIG. 44). In some embodiments, the present disclosure provides a composition or pharmaceutical composition comprising the present megakaryocytes which 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), members of the CXC chemokine family CXCL1 (GRO alfa) or CXCL12 (SDF-1), members of the TNF superfamily sCD40L or TRAIL, or members of the CC chemokine family CCL5 (RANTES), CCL11 (Eotaxin-1), CCL21 (6CKine) or CCL24 (Eotaxin-2).In some embodiments, the present disclosure provides a composition or pharmaceutical composition comprising a lysate of present megakaryocytes. These lysates can be prepared by any methods known in the art, such as membrane breakdown of preMKs or MKs by viral, enzymatic or osmotic mechanisms. Petition 870260066603, dated 06 / 07 / 2026, page 86 / 211 72 / 97 compromise its integrity. Lysates, in some embodiments, may include additional agents or be prepared in different compositions (liquid, paste, etc.), depending on the needs of specific applications. In some embodiments, these compositions may include factors such as platelet-derived growth factor isoforms PDGF-AA or PDGFBB, 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), members of the CXC chemokine family CXCL1 (GRO alpha) or CXCL12 (SDF-1), members of the TNF superfamily sCD40L or TRAIL, or members of the CC chemokine family CCL5 (RANTES), CCL11 (Eotaxin-1), CCL21 (6CKine) or CCL24 (Eotaxin-2). Methods of Use

[00157] In some embodiments, the present preMKs and MKs and their components may be a source of growth factors, such as human growth factors. In some embodiments, this growth factor may be used for cell culture, tissue regeneration, wound healing, bone regeneration, cosmeceuticals, and hemostatic bandages. In some embodiments, the present megakaryocytes or their Petition 870260066603, dated 06 / 07 / 2026, page 87 / 211 73 / 97 lysate or compositions thereof can be used in cell culture. In some embodiments, the present megakaryocytes or their lysate or compositions thereof can be used as a cosmeceutical. In some embodiments, the present megakaryocytes or their lysate or compositions thereof can be used as a therapeutic agent. For example, the present megakaryocytes or their lysate or compositions thereof can be used to increase cell expansion ex vivo, to improve bone marrow regeneration in vivo, to increase tissue regeneration and vascularization, and to increase animal survival rates in radiation studies.

[00158] In some embodiments, the present preMKs 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 in the United States and represent a large potential market for MK and resulting products as a radiation countermeasure. Exposure to radiation, such as would occur after a nuclear accident or attack, inhibits platelet production. A major radiological event would trigger an immediate demand for platelets that would deplete the existing local supply to treat emergency trauma, followed by a sustained demand for platelets. Petition 870260066603, dated 06 / 07 / 2026, page 88 / 211 4 / 97 in survivors 6+ days after exposure. National Strategic Reserve Platelet Loads will become very important as the preparedness gap for our military shifts from the front lines to 24-48 hours after the incident, when affected populations become thrombocytopenic. The United States does not maintain a platelet load in the National Strategic Reserve Load, and there are no licensed drugs that immediately increase platelet counts. Pre-MKs and Mks, according to some embodiments, can be used for on-demand platelet production. The ability to store pre-MKs for an extended period and develop on-demand hiPSC platelet production capabilities will allow the establishment of a national strategic reserve load of hiPSC platelets that will be critical to meeting this projected need.

[00159] It has been demonstrated that media supplemented with autologous platelet-rich plasma (PRP) nourishes microvascular endothelial cells to improve the preservation of vascular integrity in organs perfused for transplantation. Platelets store bioactive factors in secretory granules, which they acquire from megakaryocytes. The content includes various chemokines and growth factors, such as platelet-derived growth factor isoforms (PDGF-AA, -AB and -BB), transforming growth factor-β. Petition 870260066603, dated 06 / 07 / 2026, pages 89 / 211 75 / 97 (TGF-β), 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, -4, and -6 (BMP-2, -4, -6). Human platelet lysate dramatically increases cell expansion ex vivo, improves bone marrow regeneration in vivo, and increases animal survival rates in radiation studies.In some embodiments, the present disclosure provides a composition or pharmaceutical composition comprising a lysate of a proplatelet, preplatelet or platelet generated from the present megakaryocytes, wherein these 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), members of the CXC chemokine family CXCL1 (GRO alpha) or CXCL12 (SDF-1), members of the TNF superfamily sCD40L or TRAIL, or members of the CC chemokine family CCL5 (RANTES), CCL11 (Eotaxin-1), CCL21 (6CKine) or CCL24 (Eotaxin-2). Petition 870260066603, dated 06 / 07 / 2026, pages 90 / 211 6 / 97 Kits

[00160] The disclosure provides kits comprising a megakaryocyte or differentiated cell from the disclosure. In one embodiment, the kit includes a composition comprising an isolated megakaryocyte. In particular embodiments, the disclosure provides kits for differentiating, culturing, and / or isolating a megakaryocyte from the disclosure or a precursor thereof. In certain embodiments, the disclosure provides kits for the production of platelets.

[00161] In some embodiments, the kit comprises a sterile container containing a cellular composition; such containers may be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, foil, metal foil, or other materials suitable for containing medicinal products.

[00162] If desired, the kit is supplied with instructions for generating megakaryocytes. The instructions will generally include information on the conditions and factors necessary to differentiate, culture, and / or isolate megakaryocytes or their precursors. In some embodiments, instructions for platelet production are included. The instructions may be printed directly on the container (when present), or as a Petition 870260066603, dated 06 / 07 / 2026, pages 91 / 211 77 / 97 label applied to the container, or as a separate sheet, leaflet, card or folder supplied on or with the container.

[00163] Unless otherwise indicated, the practice described herein employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the reach of a person skilled in the art. These techniques are fully explained in the literature, such as, “Molecular Cloning: A 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 Reaction” (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides for dissemination and, as such, can be considered in the design and practice of dissemination.Techniques that are particularly useful for specific forms of implementation will be discussed in the following sections.

[00164] The following examples are presented in order to provide those skilled in the art with a disclosure and a Petition 870260066603, dated 06 / 07 / 2026, page 92 / 211 78 / 97 complete description of how to perform and use the assay, screening and therapeutic methods of disclosure and are not intended to limit the scope of the same which the inventors consider their disclosure. EXAMPLES Example 1. Extended production of megakaryocytic progenitors, megakaryocytes, and platelets from clinical-grade hiPSC cell lines.

[00165] Clinical-grade hiPSC cell lines were studied for their potential to differentiate into megakaryocytes using the targeted differentiation protocol in FIG. 2, which is a diagram showing the time course of differentiation of pluripotent stem cells into megakaryocytes, with each differentiation phase (Phase 0, I, II, III) indicated. Cell types and cell markers in Phases 0-III are represented above the timeline. Culture conditions, including medium composition, matrix, temperature, and gas conditions, are shown below the timeline.

[00166] Different iPSC cell lines are functionally distinct, and to truly optimize a differentiation protocol, it is important to identify the ideal clinical-grade cell line for the process. Three clinical-grade hiPSC cell lines were obtained, named PBG1, PBG2, and PBG3. PBG1 was obtained from NINDS Human Cell and Data. Petition 870260066603, dated 06 / 07 / 2026, p. 93 / 211 79 / 97 Repository (NHCDR) depository at NINDS (National Institute of Neurological Disorders and Stroke) / NIH (National Institutes of Health). PBG1 (NINDS ID: LiPSC-Gr1.1) was derived from CD34+ male umbilical cord blood (Lonza). PBG2 and PBG3 were obtained from Fujifilm-Cellular Dynamics International (F-CDI). PBG2 and PBG3 were derived from adult male and female blood cells, respectively (F-CDI MyCells iPSC ID numbers: 21525 and 21526).

[00167] Before the onset of differentiation, all three hiPSC cell lines formed characteristic colonies when cultured in Vitronectin with Essential 8 medium (FIG. 3A) and showed expression of the pluripotency biomarkers OCT4 and NANOG (FIG. 3B, FIG. 13A). The three clinical-grade hiPSC cell lines were directed to differentiate into megakaryocytes using a multiphase protocol summarized in FIG. 2. Cells were observed at various time points during directed differentiation (FIGS. 4A-4D). Differentiation efficiency during Phase I was initially observed by monitoring the expression of CD31+, a marker of hemogenic endothelium (FIG. 5). Differentiation efficiency was similar for 3 hiPSC cell lines during Phase I. Without being limited by theory, the differences seen in megakaryocyte production / quality later in the differentiation process did not always correlate. Petition 870260066603, dated 06 / 07 / 2026, pp. 94 / 21180 / 97 with the differentiation efficiency in Phase I. In Phase II, further differentiation toward megakaryocytic progenitors was observed by monitoring the production of CD41+CD43+ cells (FIG. 6A). Notably, for some of the clinical-grade cell lines, the production of megakaryocytic progenitors during Phase II extended for a longer time (up to 17 days) compared to previous methods using different hiPSC cell lines (FIG. 6B). Cumulative yields were especially robust for PBG-1 and PBG-2 iPSCs and showed some variability between independent differentiations (FIG. 6C). When the cells were transitioned to Phase III of the targeted differentiation protocol, CD42b+ expression increased throughout Days 1-5, and by Days 4-5 the percentage of CD41+ cells expressing CD42b+ was at least about 80% (FIG. 7A).Phase III cells obtained from the 3 clinical-grade iPSC lines were analyzed by light microscopy, immunofluorescence microscopy, and electron microscopy, which revealed that PBG1 and PBG2 exhibited characteristics consistent with those of mature megakaryocytes, including size, morphology, proplatelet extensions, megakaryocyte-specific protein expression, and ultrastructure (FIGS. 7B-7C, FIGS. 8A-8E). Example 2. Large-scale expansion of pluripotent PBG-1 cells. Petition 870260066603, dated 06 / 07 / 2026, pages 95 / 211 81 / 97

[00168] Prior to differentiation, pluripotent expansion of PBG-1 is necessary to produce the large number of cells required for a high-density seed bank, as well as to generate sufficient cell numbers to initiate differentiation on a scale appropriate for clinical production. PBG-1 can be maintained and expanded in 2D cultures using recombinant vitronectin (VTN), in addition to free animal component (ACF), cGMP-compatible reagents such as Essential 8, NutriStem, or StemFlex. Characteristic colony growth and maintenance of pluripotency markers were observed for all three growth conditions (FIGS. 9A-9C, FIGS. 10A-10C). To enable large-scale expansion, PBG-1 cells were harvested from 2D cultures as individual cells using TrypLE and allowed autoaggregation in agitated 3D vessels, in this case a 300ml DasBOX mini bioreactor system.During the first 24 hours, a ROCK inhibitor, such as Y27632, was added to the cells to promote cell survival during initial aggregation. Over 6-7 days in a shake tank, the resulting spheroids increased their diameter from 50 to 250 microns, and the total cell density increased up to 40 times within that time period (FIGS. 11A-11C). PBG-1 cells cultured in this way can be repeatedly processed and can maintain their pluripotency for at least 4 rounds. Petition 870260066603, dated 06 / 07 / 2026, page 96 / 211 82 / 97 consecutive expansions (FIGS. 12A-12B, FIG. 13B) and maintain a normal karyotype (FIG. 14). Example 3. In-depth characterization of the directed differentiation of PBG-1 to preMKs and MKs using a Collagen IV matrix in 2D culture vessels.

[00169] When harvested with 0.5 mM EDTA and divided into plates as small clusters in 4.2 µg / cm2 human Collagen IV, PBG-1 cells exhibit a characteristic set of morphological changes over 6 days of Phase I differentiation (FIG. 15). At the end of Phase I, a representative well is harvested as single cells using Accutase and evaluated by flow cytometry for the hemogenic endothelial markers CD31 and CD34 (FIG. 16A). Over multiple independent PBG-1 differentiations (n ​​= 41), the mean differentiation efficiency on day 6 was determined to be approximately 40% CD31+ (range: ~20-60%) and approximately 30% CD31+CD34+ (range ~15%-45%) (FIG. 16B).

[00170] Within 2-3 days after the start of Phase II (i.e., day 6+2 to 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 (FIG. 17A). These released cells contain preMKs, as defined by cell surface expression of CD43. Petition 870260066603, dated 06 / 07 / 2026, page 97 / 211 83 / 97 and CD41 and no CD14 expression (FIG. 17B, FIG. 17C). These floating, weakly bound Phase II cells that appear on top of the adherent cell layer are harvested daily by gentle rinsing and collection of the medium into conical tubes, and are 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 subsequent plateaus, with a peak mean preMK purity of 50-60% on day 6+6 (FIG. 18A). CD14+ myeloid cells are not major contaminants in PBG1-directed differentiation cultures during the first 6-7 days of Phase II, although there is some variability thereafter (FIG. 18B). The kinetics of preMK production peak on days 6+6 and 6+7, on average, and decrease thereafter (Fig. 19A).Across multiple independent PBG-1 differentiations (n ​​= 41), the average cumulative yield of preMK (CD43+CD41+CD14-) was determined to be approximately 1 million per well (range: 0.1 to 3.3 million) (FIG. 19B).

[00171] When preMKs from these cultures are transferred to Phase III conditions, they differentiate into mature MKs within a few days. Cells that are initially uniformly small, round, and breakable (FIG. 20A) begin to increase in size on days 2-4 (FIG. 20B and FIG. 20C). Simultaneously, proplatelet-producing MKs can be easily observed (FIG. 20C and Petition 870260066603, dated 06 / 07 / 2026, pages 98 / 211 84 / 97 FIG. 20D). By 3-4 days of Phase III, the proportion of CD61+ cells (megakaryocytic lineage) co-expressing mature MK markers CD42a and CD42b is determined by FACS (FIG. 21A and FIG. 21B) and the purity of mature MKs (CD61+CD42a+CD42b+ cells) can reach levels as high as 70-90% of all nucleated cells in the culture (FIG. 21C). Example 4. Recombinant Laminin 521 can replace Collagen IV to support targeted differentiation of PBG-1 into MKs.

[00172] Collagen IV is purified from human placental material and is only available as a reagent for research use. Therefore, collagen IV is not compatible with cGMP production from PBG1-derived megakaryocytes, and an alternative strategy must be developed before these cells can be employed for clinical use. A potential solution is to replace Collagen IV with a recombinant matrix component that is produced from recombinant sources. In this document, it is shown that recombinant Laminin-521 can be used as a suitable alternative to Collagen IV for the targeted differentiation of iPSCs into MKs. PBG-1 clusters generated by harvesting with 0.5 mM EDTA exhibited the same characteristic set of morphological changes over 6 days of Phase I differentiation in 0.13 ug / cm2 recombinant Laminin-521 as in 4.2 ug / cm2 human Collagen IV (FIGS. Petition 870260066603, dated 06 / 07 / 2026, pages 99 / 211 85 / 97 (Figs. 22A-22B). When transitioning to Phase II, Laminin-521 cultures produced similar preMK yields and purities to the corresponding Collagen IV cultures (Figs. 23A-23C). After 3 days of further differentiation, Phase III cells showed similar size, morphology, and propensity for proplatelet production, regardless of whether they were generated in Collagen IV or Laminin-521 (Figs. 24A-24B). The proportion of CD61+ cells (megakaryocytic lineage) co-expressing the mature MK markers CD42a and CD42b was also measured and found to be similar for cells generated in either matrix (Figs. 25A-25B). Example 5. WNT modulators can affect the differentiation efficiency of Phases I and II.

[00173] WNT signaling is important during development. GSK3 kinase inhibitors CHIR98014 and CHIR99021 act as WNT agonists. When the Phase I differentiation conditions described in this document (using a Laminin 521 matrix) were enhanced with CHIR98014 0.6 µM or CHIR99021 6 µM only for the first 48 hours of differentiation, a dramatic increase in Phase I differentiation efficiency was observed on day 6, as determined by CD31 and CD34 immunofluorescence staining (FIGS. 26A-26C). Control and CHIR98014 cultures were then transferred to Phase II, where preMK production and release were tracked by staining for Petition 870260066603, dated 06 / 07 / 2026, pages 100 / 211 86 / 97 immunofluorescence of CD41 and CD43. Visual estimation of the number of CD41+ cells suggests that the greater Phase I efficiency engendered by WNT modulators in the first 48 hours may correspond to a greater output during Phase II (FIGS. 27A-27B). Therefore, a short period of WNT modulator addition may affect differentiation efficiency throughout subsequent differentiation phases. Example 6. Packed bed bioreactor with macrocarriers coated with Laminin 521.

[00174] To enable the yields required for clinical production of megakaryocytes and platelets, it is crucial to transition the entire process of differentiating plastic material for small-scale tissue culture (2D, matrix-dependent) to a scalable 3D solution. In this document, we provide evidence that a Laminin 521-coated PTFE macrocarrier in the form of a 1 mm Raschig ring can support the differentiation of PBG-1 cells and that this macrocarrier material would be usable in a packed bed bioreactor, as illustrated in the scheme (FIG. 28). The PTFE rings were first incubated overnight in an oscillator at 4 °C with 1.25 μg / ml of Laminin-521. Before use, the PTFE rings were equilibrated in a 6-well plate with Essential 8 medium plus H1152, a ROCK inhibitor. Pluripotent PBG-1 iPSCs were harvested using 0.5% EDTA. Petition 870260066603, dated 06 / 07 / 2026, pages 101 / 211 87 / 97 mM, Essential 8 medium again in suspension plus H1152, and seeded as clumps in PTFE rings. Every 10 minutes, the plate was run for 30 seconds at 75 rpm on an orbital shaker. After 1 hour, the plate was continuously shaken at 75 rpm overnight. 24 hours later, 90% of the medium was removed and replaced with Phase I medium, with daily medium changes. During Phase I, PBG-1 cells exhibited growth areas within Raschig rings (FIG. 29), and the growth areas developed morphological characteristics similar to those observed in 2D cultures (FIG. 22). Flow cytometry analysis of these cells indicated a high proportion of hemogenic endothelial cells, with ~80% of the cells expressing CD31, with more than half of these cells being double-positive for CD34+ (FIG. 31A).Upon transitioning to the middle of Phase II and initiating half of the daily medium changes, the morphology shifted from a generally flat colony to a 3D spheroid-like structure, although it should be noted that these structures were still attached to the Laminin 521 coating within the ring-shaped macrocarrier (FIG. 30). Cells released during Phase II had a high preMK content, even as early as Day 6+2, with ~75% of cells co-expressing CD43 and CD41 (FIG. 31B), a purity that compares favorably to 2D matrix-dependent cultures (Fig. 18A). Cells released on Day 6+3 were... Petition 870260066603, dated 06 / 07 / 2026, pages 102 / 211 88 / 97 cells were collected and cultured for an additional 3 days in Phase III medium on an ultra-low adhesion plate, and ~80% of these cells co-expressed CD61 and CD42b (FIG. 31C), indicating that efficient MK differentiation occurred. These macrocarriers are amenable to use as material for a packed bed bioreactor in which the initial differentiation of iPSCs to hemogenic endothelium (i.e., Phase I targeted differentiation), as well as further differentiation and release of preMKs (i.e., Phase II targeted differentiation) could occur in the same vessel (FIG. 28). In this design, a packed bed bioreactor is set up with Laminin-521-coated macrocarriers freshly seeded with pluripotent PBG-1 iPSCs. The packed bed is then exposed to a continuous flow of medium to allow Phase I differentiation to hemogenic endothelium.After percolating through the packed bed, the medium would be circulated through a conditioning chamber where newly prepared medium components would be added, and oxygen / CO2 concentrations would be adjusted by spraying or other means before the medium was recirculated to the cells. At the conclusion of Phase I, the medium would be changed to allow for Phase II differentiation and the production and release of preMKs. Suitablely sized and shaped macrocarrier substrates, such as 1 mm Raschig rings, would allow for medium flow. Petition 870260066603, dated 06 / 07 / 2026, pages 103 / 211 89 / 97 channel width sufficient to allow released cells to percolate through the packed bed and out of the reactor for collection and cryostorage. This design reduces the shear forces experienced by the cells, allows for efficient use of the medium due to its perfusion-based design, and enables continuous collection of preMKs as they are released. Example 7. Self-aggregating iPSC-derived spheroids in a stirred-tank bioreactor.

[00175] Another example of a scalable 3D solution involves performing differentiations using self-aggregating spheroids suspended in agitated or shaken ultra-low adhesion vessels (FIG. 32). In this example, pluripotent PBG-1 iPSCs were dissociated into individual cells using TrypLE, resuspended at 0.51 million cells / ml in pluripotency maintenance medium (such as Essential 8, Nutristem, StemFlex, other similar media or combinations thereof) plus H1152 or another ROCK inhibitor, and incubated at 37 °C, 5% CO2, 20% O2 in a 6-well ultra-low adhesion plate on an orbital shaker at 90 rpm, or in a spin flask with constant shaking (90 rpm for a volume of 50 ml in a 125 ml spin flask). Within 24 hours in any system, PBG-1 cells self-aggregated to form spheroids approximately 50-150 µm in diameter (FIG. 33A, see also Petition 870260066603, dated 06 / 07 / 2026, pages 104 / 211 90 / 97 FIG. 11A (for example, a similar one 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 exchanged via Phase I differentiation to promote differentiation towards hemogenic endothelium, and agitation was resumed, with incubation under hypoxic conditions (37 °C, 5% CO2, 5% O2). Medium exchanges were performed similarly on a daily basis for a total of 6 days (4 days at 37 °C, 5% CO2, 5% O2, followed by 2 days at 37 °C, 5% CO2, 5% O2), during which the spheroids grew and developed the characteristic structure and shape on day 6 (FIG. 33A). When a sample of these spheroids on day 6 was dissociated and evaluated by flow cytometry, ~44% of the cells were found to express the hemogenic endothelial markers CD31 and CD34 (FIG. 33B), a purity that compares favorably to 2D matrix-dependent cultures (FIG. 16B).For the transition to Phase II, agitation was stopped and the spheroids were allowed to settle at the bottom of the vessel (approximately 5 minutes). 50-100% of the medium was then exchanged via Phase II differentiation to promote differentiation and release of cells in suspension (FIG. 34A). Subsequently, cells in suspension were collected daily and a partial medium exchange was performed. For this, agitation was stopped and the... Petition 870260066603, dated 06 / 07 / 2026, pages 105 / 211 91 / 97 hemogenic endothelial spheroids were sedimented at the bottom of the vessel (approximately 5 minutes). Approximately 80% of the medium (along with the cells in suspension) was collected and centrifuged. Half the working volume of the freshly prepared Phase II differentiation medium was added to the spheroids, along with a sufficient volume of conditioned medium (i.e., post-centrifugation supernatant) to restore the original working volume. The remaining supernatant was then discarded, with a portion of the cell bead used for FACS analysis (FIG. 34B), and the remainder cryopreserved or transferred to Phase III for maturation into mature MKs. Flow cytometry analysis of the cells in suspension revealed that most cells released between days 6+2 and 6+6 co-expressed the preMK markers CD43 and CD41 (FIG. 34B, FIG. 34C). Purities and yields of total preMK from 3D self-aggregating spheroid cultures (FIG. 34C, FIG.34D) favorably compared with purities and yields from 2D cultures (FIG. 18A, FIG. 19A). After transitioning to static Phase III cultures, preMKs from 3D self-aggregating spheroid cultures generated MK purities similar to those of preMKs from 2D culture systems (FIGS. 35A-35C). Furthermore, the Phase III differentiation cultures generated from 3D self-aggregating spheroid cultures contained cells that dramatically increased in size and were able to generate. Petition 870260066603, dated 06 / 07 / 2026, pages 106 / 211 92 / 97 proplatelets (FIG. 36), consistent with their identity as genuine megakaryocytes. Example 8. Detailed characterization of megakaryocytes derived from PBG1 iPSCs

[00176] Megakaryocytes generated using the methods described in this document demonstrate many features associated with functional mature MKs, including when converted to immunofluorescence microscopy imaging for the MK-specific beta-1-tubulin protein (FIG. 37), as well as proteins associated with alpha granules (PF4 and VWF, FIGS. 38A-38F) and dense granules (LAMP1 and serotonin, FIGS. 39A-39F). Electron microscopy images of PBG1-derived MKs reveal characteristic ultrastructural features, including multivesicular bodies, glycogen granules, and an invaginated membrane system (FIGS. 40A-40D). Gene expression analysis revealed downregulation of pluripotency genes, such as OCT4 (FIG. 41A), and upregulation of megakaryocyte lineage genes, such as NFE2 (FIG. 41B).Similar analyses were 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 (FIG. 41C).

[00177] When compared with primary megakaryocytes (natural product) derived from CD34+ cells of Petition 870260066603, dated 06 / 07 / 2026, pages 107 / 211 93 / 97 bone marrow, peripheral blood CD34+, or umbilical cord CD34+, PBG1-derived MKs, iPSC PBG1-derived MKs were found to have a similar average size (FIGS. 42A-42C), yet a characteristically lower ploidy distribution (FIGS. 43A-43B), compared to primary megakaryocytes (natural product) derived from bone marrow CD34+ cells, peripheral blood CD34+ cells, or umbilical cord stem cells (FIG. 42C, FIG. 43B). iPSC PBG1-derived megakaryocytes also had a characteristic growth factor, cytokine, and chemokine expression profile similar to those present in human platelets, including the presence of multiple factors not previously reported in megakaryocytes (FIG. 44). To prepare the data, hiPSC-MK cells at 25 million / mL in 1X PBS were lysed by freezing the cells at -80 °C overnight and then thawing at 37 °C. This freeze / thaw cycle was repeated 4 times.The resulting suspension was filtered using a 0.22 µm syringe filter. Lysates were tested for a selected panel of growth factors, cytokines, and chemokines using multiplexing laser bead technology (Eve Technologies). Data were corrected to reference (PBS, which was processed similarly to hiPSC-MK), then compared to commercially available human platelet lysate (HPL), medium of. Petition 870260066603, dated 06 / 07 / 2026, pages 108 / 211 94 / 97 differentiation of freshly prepared MK (used in the final differentiation stage) and conditioned medium, i.e., MK differentiation medium removed from hiPSC-MK prior to lysis. Although a strong overlap was observed between hiPSC-MK and HPL, there were also several proteins measured in hiPSC-MK that had not been previously described in megakaryocytes or platelets (as indicated by “).

[00178] The results described in this document demonstrate a robust process for the generation of clinical-grade human iPSC-derived megakaryocytes. Human iPSC-derived megakaryocytes can be isolated and concentrated for further characterization or use in downstream applications, such as the generation of human platelets (FIGS. 45A-45C). Other forms of implementation

[00179] As described in this document, the present disclosure presents compositions and methods for the production of megakaryocytic progenitors and megakaryocytes. In one aspect, the disclosure provides a megakaryocyte or megakaryocytic progenitor differentiated from a clinical-grade hiPSC cell or cell line. In some embodiments, the present disclosure provides an isolated cell population comprising the megakaryocyte or megakaryocytic progenitor according to any aspect outlined in this document. In some embodiments, the present disclosure provides Petition 870260066603, dated 06 / 07 / 2026, pages 109 / 211 95 / 97 a composition comprising a megakaryocyte or megakaryocytic progenitor according to any aspect outlined in this document.

[00180] In some embodiments, the disclosure provides a composition or pharmaceutical composition comprising a lysate of a megakaryocyte according to any aspect indicated in this document. In some embodiments, the disclosure provides a composition or pharmaceutical composition comprising a lysate of a platelet generated from the megakaryocyte according to any aspect indicated in this document. In some embodiments, the disclosure provides a method of producing a megakaryocyte, the method comprising differentiating a clinical-grade hiPSC cell or cell line. In various embodiments of any aspect indicated herein, the megakaryocyte is one or more CD42b+, CD61+, and DNA+. In various embodiments of any aspect indicated herein, the megakaryocyte has a diameter of about 10-30 µm. In certain embodiments, the megakaryocyte has a diameter of about 10-20 µm.In various embodiments of any aspect indicated in this document, the megakaryocyte has a ploidy of at least 4N, 8N, or 16N. In various embodiments of any aspect indicated in this document, the megakaryocytic progenitor is CD14-, CD41+, and CD43+. In various embodiments of any aspect. Petition 870260066603, dated 06 / 07 / 2026, pp. 110 / 211 In various embodiments of any aspect indicated in this document, the megakaryocytic progenitor is capable of being continuously produced until at least day 10 after the onset of differentiation of a hemoendothelial progenitor (i.e., Phase II, Day 6+10). In various embodiments of any aspect indicated in this document, the megakaryocytic progenitor is capable of being continuously produced until at least day 17 after the onset of differentiation of a hemoendothelial progenitor (i.e., Phase II, Day 6+17). In various embodiments of any aspect indicated in this document, the clinical-grade hiPSC cell or cell line is selected from the group consisting of PBG1, PBG2, and PBG3.

[00181] In various embodiments of any aspect indicated herein, the isolated cell population or composition contains a platelet generated from a megakaryocyte of the dissemination. In various embodiments of any aspect indicated herein, the isolated cell population or composition contains at least 50% CD42b+CD41+CD61+ cells. In various embodiments of any aspect indicated herein, the isolated cell population or composition contains at least 50% megakaryocytes with ploidy of 4N or higher. In certain embodiments, at least 50% of the megakaryocytes have ploidy of 4N-16N. In various embodiments of any aspect indicated herein, the isolated cell population or Petition 870260066603, dated 06 / 07 / 2026, pages 111 / 211 97 / 97 of the composition contains megakaryocytes with an average ploidy of 4N or higher.

[00182] From the preceding description, it will be evident that variations and modifications can be made to the disclosure described in this document to adapt it to various uses and conditions. Such embodiments are also within the scope of the following claims.

[00183] The recitation of a list of elements in any definition of a variable includes in this document definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of a realization form herein includes that realization form as any single realization form or in combination with any other realization forms or portions thereof.

[00184] All patents and publications mentioned in this descriptive report are incorporated herein by reference to the same extent as if each independent patent and publication were specifically and individually designated for incorporation by reference. Petition 870260066603, dated 06 / 07 / 2026, pp. 112 / 211

Claims

1 / 2 CLAIMS 1. A method for producing megakaryocytes, characterized in that it comprises: cultivating dissociated pluripotent stem cells in a matrix-independent culture under continuous agitation, such that the pluripotent stem cells form self-aggregating pluripotent cell spheroids; differentiating, under continuous agitation, the self-aggregating pluripotent cell spheroids in a first culture medium into hemogenic endothelial cell spheroids; differentiating, under continuous agitation, the hemogenic endothelial cell spheroids in a second culture medium to produce megakaryocytic progenitors, causing the hemogenic endothelial cell spheroids to release the megakaryocytic progenitors in suspension, while maintaining the hemogenic endothelial cell spheroids for the subsequent production and release of the megakaryocytic progenitors.

2. Method according to claim 1, characterized in that 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).

3. Method, according to claim 1, characterized in that the second culture medium Petition 870260066603, dated 06 / 07 / 2026, page 10 / 211 2 / 2 comprises one or more of Stem Cell Factor (SCF), Thrombopoietin (TPO), Fms-related tyrosine kinase ligand 3 (Flt3-L), Interleukin-3 (IL-3), Interleukin-6 (IL-6) and Heparin.

4. Method according to claim 1, characterized in that the pluripotent stem cells are human-induced pluripotent stem cells.

5. Method according to claim 1, characterized in that it further comprises the step of seeding megakaryocytic progenitors on a non-adherent surface in a culture medium before differentiating the megakaryocytic progenitors into megakaryocytes. Petition 870260066603, dated 06 / 07 / 2026, p. 11 / 211

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