Megakaryocyte derivatives for the treatment of dry eye disease

Abstract

Dry eye disease (DED) is an example of a disease with a significant unmet need and a pressing need for treatment. Unfortunately, current drug therapies are ineffective for many dry eye patients, and the disease often progresses. Therefore, there is a need for novel, less aggressive, and better treatment options for dry eye disease, as described in this application. Methods for treating, repairing, or restoring diseases, disorders, or injuries associated with dry eye disease, wound healing, and osteoarthritis are described herein. Methods for generating megakaryocyte derivatives are also described herein.

Description

【Technical Field】 【0001】 Cross - reference applications This application claims the benefit of priority based on U.S. Provisional Application No. 63 / 437,578, filed on January 6, 2023, the entire content of which is incorporated herein by reference. 【Background Art】 【0002】 Background Blood derivatives such as platelet - rich plasma (PRP), platelet - rich growth factor plasma (PRGF), and autologous serum (AS) are involved in playing a role in medical treatments for wound healing or accelerating the repair of damaged tissues such as cartilage, tendons, ligaments, and bones, or in the treatment of dry eye and other diseases. However, due to the limited number of reported data cases from platelet donors, important data regarding the beneficial effects of these blood derivatives in clinical procedures is lacking. Furthermore, platelet donors are different from each other, and thus, there is no consistency in the quality of PRP or other derivatives produced for medical procedures. Currently used autologous blood derivatives have several other disadvantages such as variability between batches because each patient provides their own blood as a source of PRP, PRGF, or AS. Other disadvantages include the short lifespan of donor platelets, impurities or contamination, or difficulty in obtaining or insufficient supply. As a result of the lack of standardization of these products, the clinical effectiveness of blood derivatives is still under discussion. Therefore, in this field, instead of having to create separate and unreliable preparations for each patient, for whom there is a skeptical view regarding their potential effectiveness and use, there is an urgent need for safe, standardized, and regulated off - the - shelf products that can be developed and approved for human use by regulatory agencies. 【0003】 Dry eye disease (DED) is an example of a disease with large unmet needs and a pressing need for treatment. In current DED treatment, patients are prescribed one of several treatments, for example, Restasis® which has the active ingredient cyclosporine A (CsA). Unfortunately, current drug therapies are not effective for many dry eye patients and the disease often progresses. Dry eye disease is classified into two categories: (i) lacrimal deficiency and (ii) evaporative. The evaporative causes of dry eye disease are due to several of insufficient oil, eyelid changes, contact lens use, or ocular surface diseases (OSD) such as allergic conjunctivitis, and iatrogenic dry eye that occurs after the use of systemic or topical drug therapy, or after surgery or non-surgical procedures. If symptoms continue to progress, the patient is determined to need surgery and is referred to an ophthalmologist. Accordingly, there is a need for new, less aggressive, and better treatment options for the treatment of dry eye disease, as described in this application. Summary of the Invention Means for Solving the Problems 【0004】 Abstract A method of treating, repairing or restoring dry eye disease in a subject that requires treatment of dry eye disease is provided herein. In some embodiments, the method comprises administering to the subject an effective amount of a composition comprising a megakaryocyte derivative, wherein the route of administration is ocular or intra-glandular, and the megakaryocyte derivative comprises a megakaryocyte-like cell (MLC) lysate, a platelet-like cell (PLC), or a combination thereof. 【0005】 Provided herein are methods for treating, repairing, or restoring a condition in a subject requiring treatment of the condition, comprising administering to the subject multiple doses of an effective amount of a composition containing a megakaryocyte derivative, wherein the condition is selected from dry eye disease, ocular surface disease, osteoarthritis, and wound healing, and the megakaryocyte derivative comprises MLC lysate, PLC, exosomes, megakaryocytes, or a combination thereof, and the megakaryocyte derivative is derived from fibroblast growth factor-2 (FGF-2); hepatocyte growth factor (HGF); insulin-like growth factor 1 (IGF-1); activated regulated normal T cell expression secretion factor (RANTES); neurotrophic growth factor (NGF); vascular endothelial growth factor (VEGF-A); vascular endothelial growth factor (VEGF-C); epidermal growth factor (EGF). The biomarker comprises one or more biomarkers selected from transforming growth factor-β1 (TGF-β1); transforming growth factor-β2 (TGF-β1); platelet-derived growth factor-AA (PDGF-AA); platelet-derived growth factor-BB (PDGF-BB); platelet-derived growth factor-AA / BB (PDGF-AA / BB); interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-12p40 (IL-12p40); interleukin-12p70 (IL-12p70); tissue metalloproteinase inhibitor 1 (TIMP-1); tissue metalloproteinase inhibitor 2 (TIMP-2); and tissue metalloproteinase inhibitor 3 (TIMP-3). 【0006】 A method for producing megakaryocyte derivatives is provided herein, comprising: culturing a population of progenitor cells ex vivo for a period of time for the progenitor cells to differentiate into mature megakaryocytes; isolating a population of MLCs, PLCs, or their derivatives by separating them from megakaryocytes; concentrating the MLCs, PLCs, or their derivatives; optionally lysing the MLCs, PLCs, or their derivatives; optionally mixing them with donor-derived PRP, wherein the megakaryocyte derivative comprises MLC lysates, PLCs, exosomes, megakaryocytes, or a combination thereof. 【0007】 In some embodiments, the techniques described herein relate to methods for treating, repairing or restoring a condition in a subject requiring such treatment, comprising administering to the subject an effective amount of a composition comprising a megakaryocyte derivative, wherein the condition is a dry eye disease, the route of administration is ophthalmos or intraglandular, and the megakaryocyte derivative comprises megakaryocyte-like cell (MLC) lysates, platelet-like cells (PLC), or a combination thereof. 【0008】 In some embodiments, the techniques described herein relate to methods in which the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, a neurotropic agent, an anti-hormone agent, or an immunomodulator, or a combination thereof. 【0009】 In some embodiments, the techniques described herein relate to a method in which the tissue regeneration agent is (a) a growth factor selected from one or more of the following: transforming growth factor (TGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet-derived endothelial growth factor (PDEGF), platelet-derived angiogenic factor (PDAF), platelet factor 4 (PF-4), hepatocyte growth factor (HGF), or a combination thereof; and (b) one or more of the following cytokines selected from one or more of the following: IL-1B, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17A, IL-23, TNF-alpha, or a combination thereof. 【0010】 In some embodiments, the techniques described herein involve megakaryocyte derivatives such as fibroblast growth factor-2 (FGF-2), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), activated regulated normal T cell expression secretion factor (RANTES), neuronal growth factor (NGF), vascular endothelial growth factor (VEGF-A), vascular endothelial growth factor (VEGF-C), epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), transforming growth factor-β2 (TGF-β1), and platelet-derived growth factor-AA (PDGF-AA). The present invention relates to a method comprising one or more biomarkers selected from platelet-derived growth factor-BB (PDGF-BB); platelet-derived growth factor-AA / BB (PDGF-AA / BB); interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-12p40 (IL-12p40); interleukin-12p70 (IL-12p70); tissue metalloproteinase inhibitor 1 (TIMP-1); tissue metalloproteinase inhibitor 2 (TIMP-2); and tissue metalloproteinase inhibitor 3 (TIMP-3). 【0011】 In some embodiments, the techniques described herein relate to a method in which the megakaryocyte derivative contains an EGF concentration per unit total protein that is higher than the mean EGF concentration per unit total protein measured in human platelets, platelet-rich plasma (PRP), or multi-growth factor plasma (PRGF). 【0012】 In some embodiments, the techniques described herein relate to methods for producing megakaryocyte derivatives from cells derived from the reprogramming and / or differentiation of somatic cells, progenitor cells, or stem cells. 【0013】 In some embodiments, the techniques described herein relate to methods in which the megakaryocyte derivative is not a cancerous cell. 【0014】 In some embodiments, the techniques described herein relate to methods for which megakaryocyte derivatives do not exhibit uncontrolled proliferation or tumorigenesis in vivo. 【0015】 In some embodiments, the techniques described herein relate to methods for reducing injury recovery time. 【0016】 In some embodiments, the techniques described herein relate to methods for improving one or more signs and / or symptoms of dry eye disease. 【0017】 In some embodiments, the techniques described herein relate to methods in which the composition further comprises a lysate, serum, plasma, multi-growth factor plasma (PRGF), platelet-rich plasma (PRP), or any other blood derivative derived from the subject. 【0018】 In some embodiments, the techniques described herein relate to methods in which the route of administration of eye drops is subconjunctival or topical. 【0019】 In some embodiments, the techniques described herein relate to methods in which the administration route of eye drops is under Tenon's. 【0020】 In some embodiments, the techniques described herein relate to methods in which the route of administration of eye drops is intravitreous or intrachorally. 【0021】 In some embodiments, the techniques described herein relate to methods for treating dry eye disease caused by Sjögren's syndrome or non-Sjögren's syndrome. 【0022】 In some embodiments, the techniques described herein relate to methods in which the megakaryocyte derivative does not contain red blood cells or hemoglobin contents or white blood cells. 【0023】 In some embodiments, the techniques described herein relate to methods in which the composition further comprises extracellular vesicles (EVs). 【0024】 In some embodiments, the techniques described herein relate to methods for which a composition is formulated for application to a site of injury or tissue damage for therapeutic use. 【0025】 In some embodiments, the techniques described herein relate to methods in which a composition is formulated in a buffer, diluent, or excipient, or a combination thereof. 【0026】 In some embodiments, the techniques described herein relate to a method in which the composition comprises a) 0.01 to 100% by weight of a megakaryocyte derivative, b) 0 to 90% by weight of an expander, and / or c) 0 to 90% by weight of at least one excipient or carrier, and optionally d) further comprising platelet-rich plasma (PRP), PRGF, plasma, serum, or other blood derivatives derived from the subject. 【0027】 In some embodiments, the techniques described herein relate to methods by which the composition is freeze-dried. 【0028】 In some embodiments, the techniques described herein relate to methods in which a composition is embedded in an implantable device. 【0029】 In some embodiments, the techniques described herein relate to methods for which compositions are cryopreserved. 【0030】 In some embodiments, the techniques described herein relate to a method by which a composition is administered topically to one or more sites of injury or disease or its vicinity. 【0031】 In some embodiments, the techniques described herein relate to methods in which the composition further comprises another therapeutic agent. 【0032】 In some embodiments, the techniques described herein are methods for treating, repairing, or restoring a condition in a subject requiring such treatment, comprising administering to the subject multiple doses of an effective amount of a composition comprising a megakaryocyte derivative, wherein the condition is selected from dry eye disease, ocular surface disease, osteoarthritis, and wound healing, and the megakaryocyte derivative comprises MLC lysate, PLC, exosomes, megakaryocytes, or a combination thereof, and the megakaryocyte derivative comprises fibroblast growth factor-2 (FGF-2); hepatocyte growth factor (HGF); insulin-like growth factor 1 (IGF-1), activated regulated normal T cell expression secretion factor (RANTES); neurotrophic growth factor (NGF); vascular endothelial growth factor (VEGF-A); vascular endothelial growth factor (VEGF-C); epidermal growth factor The present invention relates to a method comprising one or more biomarkers selected from (EGF); transforming growth factor-β1 (TGF-β1); transforming growth factor-β2 (TGF-β1); platelet-derived growth factor-AA (PDGF-AA); platelet-derived growth factor-BB (PDGF-BB); platelet-derived growth factor-AA / BB (PDGF-AA / BB); interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-12p40 (IL-12p40); interleukin-12p70 (IL-12p70); tissue metalloproteinase inhibitor 1 (TIMP-1); tissue metalloproteinase inhibitor 2 (TIMP-2); and tissue metalloproteinase inhibitor 3 (TIMP-3). 【0033】 In some embodiments, the techniques described herein relate to methods in which the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, a neurotropic agent, an anti-hormone agent, or an immunomodulator, or a combination thereof. 【0034】 In some embodiments, the techniques described herein relate to methods in which multiple doses are administered daily, weekly, bi-weekly, every three weeks, or monthly. 【0035】 In some embodiments, the techniques described herein relate to methods by which a composition is administered via one of the following routes of administration: topical, transdermal, or systemic. 【0036】 In some embodiments, the techniques described herein relate to a method by which a composition is administered by one of the following routes of administration selected from subconjunctival, sub-Tenon, intravitreous, anterior chamber, intravenous, intra-arterial, intramuscular, subcutaneous, inhalation, rectal, oral, vaginal, intraperitoneal, intra-articular, intraglandular, topical, ear, or oral administration routes. 【0037】 In some embodiments, the techniques described herein relate to methods in which the condition is a dry eye disease, the treatment is not for cancer, and the route of administration is by eye drops, intraglandular, subconjunctival, topical, sub-Tenon's, intravitreous, or anterior chamber. 【0038】 In some embodiments, the techniques described herein relate to methods in which a composition is diluted to a physiological concentration in a carrier, and the carrier contains a diluent or excipient. 【0039】 In some embodiments, the techniques described herein relate to methods in which the carrier is plasma or plasma substitute or plasma light or physiological saline. 【0040】 In some embodiments, the techniques described herein are methods for producing megakaryocyte derivatives, comprising: culturing a population of progenitor cells ex vivo for a period of time for the progenitor cells to differentiate into mature megakaryocytes; isolating a population of MLCs, PLCs, or their derivatives by separating them from megakaryocytes; concentrating the MLCs, PLCs, or their derivatives; optionally lysing the MLCs, PLCs, or their derivatives; optionally mixing them with donor-derived PRP, wherein the megakaryocyte derivatives comprise MLC lysates, PLCs, exosomes, megakaryocytes, or combinations thereof. 【0041】 In some embodiments, the techniques described herein involve progenitor cells such as human induced pluripotent stem cells (iPSCs), hematopoietic stem cells, embryonic stem cells (ESCs), immortalized megakaryocyte progenitor cells, and CD34. + Umbilical cord blood stem cells (UCB cells), CD34 + Mobilized peripheral blood cells (MPB cells) or CD34 + The present invention relates to a method of selecting one or more bone marrow cells. 【0042】 In some embodiments, the techniques described herein relate to compositions for treating dry eye disease in a subject, and such compositions are prepared according to one of the methods provided herein. 【0043】 In some embodiments, the techniques described herein involve megakaryocyte derivatives such as fibroblast growth factor-2 (FGF-2), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), activated regulated normal T cell expression secretion factor (RANTES), neuronal growth factor (NGF), vascular endothelial growth factor (VEGF-A), vascular endothelial growth factor (VEGF-C), epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), transforming growth factor-β2 (TGF-β1), platelet-derived growth factor-AA (PDGF-AA), and blood The present invention relates to a composition comprising one or more biomarkers selected from platelet-derived growth factor-BB (PDGF-BB); platelet-derived growth factor-AA / BB (PDGF-AA / BB); interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-12p40 (IL-12p40); interleukin-12p70 (IL-12p70); tissue metalloproteinase inhibitor 1 (TIMP-1); tissue metalloproteinase inhibitor 2 (TIMP-2); and tissue metalloproteinase inhibitor 3 (TIMP-3). 【0044】 In some embodiments, the techniques described herein relate to compositions in which the megakaryocyte derivative contains an EGF concentration per unit total protein that is higher than the average EGF concentration per unit total protein measured in human platelets, platelet-rich plasma (PRP), or multi-growth factor plasma (PRGF). 【0045】 In some embodiments, the techniques described herein further relate to compositions comprising wound healing agents, tissue regeneration agents, anti-apoptotic agents, anti-inflammatory agents, neurotropic agents, anti-hormone agents or immunomodulators, or combinations thereof. 【0046】 In some embodiments, the techniques described herein relate to compositions in which the megakaryocyte derivative does not contain red blood cells or hemoglobin contents or white blood cells. 【0047】 In some embodiments, the techniques described herein relate to compositions further comprising extracellular vesicles (EVs). 【0048】 In some embodiments, the techniques described herein relate to compositions formulated for application to a site of injury or tissue damage for therapeutic use. 【0049】 In some embodiments, the techniques described herein relate to compositions further comprising another therapeutic agent. 【0050】 In some embodiments, the techniques described herein relate to any one of the methods provided herein by which megakaryocyte derivatives are fabricated in a fluid device or a bioreactor. 【0051】 This disclosure is described in the following detailed description with reference to several drawings described herein, by non-limiting examples of exemplary embodiments, where similar reference figures represent similar parts across several figures of the drawings. [Brief explanation of the drawing] 【0052】 [Figure 1A-1]Figures 1A-1C show the growth factor and cytokine profiles in PLC compared to donor PRP and washed donor platelets. [Figure 1A-2] Same as above. [Figure 1A-3] Same as above. [Figure 1A-4] Same as above. [Figure 1B-1] Same as above. [Figure 1B-2] Same as above. [Figure 1C-1] Same as above. [Figure 1C-2] Same as above. [Figure 1C-3] Same as above. 【0053】 [Figure 2A] Figure 2A shows a schematic design of a PLC treatment study for dry eye disease in mice. Figures 2B-2C show the results of corneal fluorescein staining and phenol red thread tests, respectively, to evaluate mice treated with PLC and induced dry eye disease, compared to cyclosporine A (CsA) as a positive control. Figure 2D shows PLC-mediated recovery of goblet cell loss in a DS-induced DED model, as measured by the difference in goblet cell density in the conjunctiva of vehicle-treated mice versus PLC-treated mice. Figure 2E shows representative histological images of PAS-stained conjunctiva of vehicle-treated mice versus PLC-treated mice. Goblet cells stain darkly along the conjunctival margin. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. 【0054】 [Figure 3] Figure 3 shows a combination of corneal fluorescein staining data from the study presented in Figure 2 and follow-up studies using the same DED model. 【0055】 [Figure 4A]Figure 4A shows a schematic design of a PLC treatment study for Sjögren's syndrome using an in vivo TSP1 knockout mouse model. Figure 4B summarizes the results for each group pooled from several studies, measured by the mean change in corneal fluorescein score for each eye, by comparing each eye 2 weeks after treatment to the same eye before treatment, at baseline (i.e., before treatment) and at endpoint (i.e., after treatment). Figure 4C shows a representative image with marked areas of corneal fluorescein staining. Figure 4D shows further time-course results from four arms of the study, where the same eye was measured again 2 weeks after treatment cessation. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. 【0056】 [Figure 5A] Figures 5A and 5B are heatmaps of profiled cytokines and growth factors, showing the relative concentrations of cytokines and growth factors measured by profiling in MLC and various MLC derivatives compared to human blood-derived PRGF, and in PLC and various MLC derivatives compared to human blood-derived PRGF, respectively. [Figure 5B] Same as above. 【0057】 [Figure 6A] Figures 6A–6D provide a comparative analysis of measured concentrations of major cytokines and other factors in MLC and various MLC derivatives compared to PRGF derived from human blood. Figure 6A shows major platelet / plasma-related factors. Figure 6B shows putative epitheliotrophic factors. Figure 6C shows putative pro-resolutory T cell factors. Figure 6D shows matrix metalloproteinase inhibitors. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D]Same as above. 【0058】 [Figure 7A] Figures 7A–7D provide a comparative analysis of measured concentrations of major cytokines and other factors in MLC and various MLC derivatives compared to PRGF derived from human blood. Figure 7A shows major platelet / plasma-related factors. Figure 7B shows putative epitheliotropic factors. Figure 7C shows putative dissipation-promoting T cell factors. Figure 7D shows matrix metalloproteinase inhibitors. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. 【0059】 [Figure 8] Figure 8A shows the total protein analysis (BCA). Figures 8B and 8C provide ELISA-based quantifications of EGF and PDGF-BB in various MLC derivatives, respectively. 【0060】 [Figure 9] Figures 9A-9B show the particle distribution and particle concentration (1750-10000 nm) for various megakaryocyte derivative preparations. 【0061】 [Figure 10] Figure 10A shows the results of the effect of EGF on HCE-T cell proliferation in a 72-hour time-course study at doses ranging from 30.4 pg / ml to 31.2 ng / ml. Figure 10B quantifies the data from 10A using the area under the curve normalized to the vehicle. 【0062】 [Figure 11-1]Figure 11A shows the results of the effect of MLC lysates on HCE-T cell proliferation in a 72-hour time-course study at doses ranging from 0.025% (v / v) to 0.75% (v / v). Figure 11B quantifies the data from 11A using the area under the curve normalized to the vehicle. Figure 11C shows the results of the effect of MLC lysates prepared by 0.22 μm filtration on HCE-T cell proliferation in a 72-hour time-course study at doses ranging from 0.0128% (v / v) to 1.0% (v / v). Figure 11D quantifies the data from 11C using the area under the curve normalized to the vehicle. [Figure 11-2] Same as above. 【0063】 [Figure 12] Figures 12A and 12B show the results of a carboxyfluorescein diacetate succinmidyl ester (CFSE)-based flow cytometry assay. Figure 12A shows the mean fluorescence intensity (MFI), with lower MFI indicating a higher growth index. Figure 12B shows the same data as a normalized ratio, with higher ratios indicating a higher growth index. 【0064】 [Figure 13-1] Figure 13A shows a schematic design of a study to evaluate the in vivo effects of megakaryocyte derivative treatment in a rat medial meniscus tear (MMT) model of osteoarthritis (OA). Figures 14B–14G show the results of the efficacy of megakaryocyte derivatives, MLC lysates, and PLC in dynamic weight-bearing tests (Figure 13B), electron von Frey filament analysis (Figure 13C), histology of MMT joints (Figure 13D), substantial cartilage degeneration width (Figure 13E), synovitis score (Figure 13F), and medial tibial osteophyte measurements (Figure 13G). [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 13-4] Same as above. 【0065】 [Figure 14]Figures 14A and 14B show the effect of PLC on HUVEC migration in the scratch assay. Figure 14A shows the time-course data (120 hours) of the obtained cell index, and Figure 14B shows the AUC data normalized to the vehicle. 【0066】 [Figure 15-1] Figure 15A shows a schematic design of a study to evaluate the in vivo effects of megakaryocyte derivatives, MLC lysates, and PLC treatment in a db / db diabetic mouse model of wound healing. Figures 15B–15F show the efficacy results of PLC and MLC lysates, including wound closure kinetics (Figure 15B), histological results of diabetic mouse wounds (Figure 15C), histological analysis of wounds for quantification of wound closure (Figure 15D), granulation tissue area (Figure 15E) and epidermal tissue area (Figure 15F), histological visualization of the endomucin-based vascular system (Figure 15G), and quantification of endomucin fluorescence signaling (Figure 15H). [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 15-4] Same as above. [Figure 15-5] Same as above. 【0067】 [Figure 16] Figure 16A shows a schematic diagram of the differentiation process. 【0068】 [Figure 17A] Figure 17A shows a schematic diagram of the PLC production process. Figure 17B shows an exemplary microfluidic bioreactor (BioR) device for processing MLC. [Figure 17B] Same as above. [Modes for carrying out the invention] 【0069】 The drawings shown above illustrate the currently disclosed embodiments, but other embodiments are also conceived, as noted in the discussion. This disclosure provides representative and exemplary embodiments, and is not limiting. Numerous other modifications and embodiments within the scope of the principles and spirit of the currently disclosed embodiments can be devised by those skilled in the art. 【0070】 Detailed explanation Unless otherwise specified, all technical and scientific terms used herein have meanings that are generally understood by those skilled in the art in which this disclosure pertains. The following references provide general definitions of many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2 nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5 th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings of those below unless otherwise specified. 【0071】 When used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is nontoxic to the exposed cells or mammals at the dose and concentration used. In many cases, a physiologically acceptable carrier is a pH-buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphates, phosphate-buffered saline (PBS), citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than approximately 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; plasma or plasma substitutes, dextran and hydroxyethyl starch, hemoglobin without perfluorocarbons and stromas; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. 【0072】 As used herein, the term "derivative" refers to differentiation into different types of cells, cells produced from such cells by genetic manipulation or genetic modification, or compositions produced from such cells (e.g., by lysing such cells). In other words, the term "derivative" encompasses any genetic, chemical modification of cells or any combination thereof. 【0073】 The term “megakaryocyte derivative” as used herein includes megakaryocytes or MLC derivatives. In some embodiments, megakaryocyte derivatives include megakaryocytes or MLCs (e.g., lysates, EVs) or any composition produced from other cells differentiated from megakaryocytes or MLCs. In some embodiments, megakaryocyte derivatives include MLCs, PLCs, their derivatives or their lysates or PRPs, PRGFs, or ASs derived therefrom. In some embodiments, megakaryocyte derivatives include compositions produced from genetically engineered MLCs (eMLCs) or PLCs (ePLCs). 【0074】 As used herein, “progenitor cells” refers to iPSC-derived cells such as pre-MK, MK, platelet precursor (proplatelet), and preplatelet. “Pluripotent stem cells” also encompass embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells, as well as other stem cells that have the ability to form cells from all three germ layers of the body, regardless of how the pluripotent stem cells are derived. Pluripotent stem cells are defined as stem cells that may functionally possess one or more of the following characteristics: (a) the ability to induce teratomas when transplanted into immunodeficient (SCID) rats; (b) the ability to differentiate into cell types of all three germ layers (e.g., ectoderm, mesoderm, and endoderm); or (c) the expression of one or more embryonic stem cell markers (e.g., expressing Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, SSEA-5 surface antigen, Nanog, TRA-1-60, TRA-1-81, SOX2, or REX1). Progenitor cells also include “megakaryotic progenitor cells” (preMK), which refer to mononuclear hematopoietic cells that are involved in the megakaryocyte lineage and are precursors to mature megakaryocytes. Megakaryotic progenitor cells are typically found in the bone marrow and other hematopoietic sites (but are not limited to these), but can also be generated from pluripotent stem cells, for example, through further differentiation of hematopoietic endothelial cells that themselves originate from pluripotent stem cells. 【0075】 The term “megakaryotic progenitor cell” (pre-MK), as used herein, refers to mononuclear hematopoietic cells that are involved in the megakaryocyte lineage and are precursors to mature megakaryocytes. Megakaryotic progenitor cells are typically found in the bone marrow and other hematopoietic sites (but are not limited to these), but can also be generated from pluripotent stem cells, for example, by further differentiation of hematopoietic endothelial cells that themselves originate from pluripotent stem cells. 【0076】 The term "induced pluripotent stem cells" (iPS cells or iPSCs) refers to a type of pluripotent stem cell produced by reprogramming somatic cells by expressing a combination of reprogramming factors. iPSCs can be produced using fetal, postnatal, neonatal, juvenile, or adult somatic cells. Factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4 (sometimes referred to as Oct3 / 4), Sox2, c-Myc, and Klf4. In some embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4, Sox2, Nanog, and Lin28. In some embodiments, at least two, three, or four reprogramming factors are expressed in somatic cells to reprogram them. 【0077】 "Agonist activation" refers to the activation of cell receptors or ligands, which is induced by receptor-specific agonists. Agonists activate cells by binding to their respective receptors or ligands on the cell. 【0078】 As used herein, "variant" refers to any structural variation, structural deviation, or structural difference. A variant also includes PLC or its derivatives or any other product produced by culturing megakaryocytes in PLC or microsomes, exosomes, vesicles, or bioreactors. 【0079】 "PLC" (singular or plural), or artificial platelets, as used interchangeably herein, refers to novel, anucleate platelets or platelet-like cells that do not occur naturally and are structurally different from naturally occurring bone marrow-derived platelets (i.e., the natural counterpart). PLC (singular or plural) also encompasses platelet variants as defined elsewhere. 【0080】 "MLC" (singular or plural), or artificial megakaryocytes, as used interchangeably herein, refers to novel megakaryocytes or megakaryocyte-like cells that do not occur naturally and are structurally different from naturally occurring bone marrow-derived megakaryocytes (i.e., the natural counterpart). 【0081】 As used herein, "variant" (singular or plural), as used interchangeably herein, refers to exhibiting structural diversity, structural deviation, or structural difference between PLC and donor platelets. As a non-limiting example, a variant has an average of more than 2% CD63 receptor, i.e., (CD63 <平均2% ) when compared to reference resting bone marrow-derived platelet cells having an average of less than 2% CD63 receptor, i.e., (CD63 >平均2% ). In some embodiments, a variant has an average of less than 10% CD36 receptor, i.e., (CD36 >平均80% ) when compared to reference resting bone marrow-derived platelet cells having an average of more than 80% CD36 receptor, i.e., (CD36 <平均80% ), or an average of less than 95% CD42b receptor, i.e., (CD42b >平均95% ) when compared to reference resting bone marrow-derived platelet cells having an average of more than 95% CD42b receptor, i.e., (CD42b <平均95% ); or a variant having an average of less than 90% or less GPVI receptor, i.e., (GPVI >平均90% ) when compared to reference resting bone marrow-derived platelet cells having an average of more than 90% glycoprotein VI receptor, i.e., (GPVI <平均90%The term "variant" also encompasses a structural configuration of PLC that, in resting or their activated phase, is comparable to the structural configuration of naturally occurring bone marrow-derived platelets. For example, PLC and donor platelets may have receptors for m%CD36, or n%CD42a, or o%CD42a-bd, or p%CD61, or q%CD62p, or x%CD63, where m%, n%, o%, p%, q%, and x% are the same (i.e., have equal values) between PLC and bone marrow-derived platelets. In other words, structurally, PLC may be identical to donor platelets, but the PLC variants disclosed herein represent advantages. 【0082】 "Containing," "containing," "containing," and "having," etc., may have meanings attributable to them in U.S. patent law, and may also mean "encompassing," "encompassing," etc. (for example, a composition "containing" X may consist solely of X or may contain further things, e.g., X + Y), and similarly, "essentially from" or "essentially from" may have meanings attributable to them in U.S. patent law, and such terms are open-ended and allow for entities beyond those enumerated, as long as the basic or novel features of those enumerated are not altered by those entities beyond those enumerated, but exclude embodiments of the prior art. 【0083】 Unless otherwise specified or evident from the context, the term “or” is understood to be inclusive as used herein. Unless otherwise specified or evident from the context, the terms “a,” “an,” and “the” are understood to be singular or plural as used herein. 【0084】 Unless otherwise specified or made clear from the context, the term “about” as used herein is understood to mean within the normal range of acceptance in the art, for example, within two standard deviations of the mean. “About” 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 made clear from the context, all numerical values ​​provided herein are modified by the term “about.” 【0085】 As used herein, “mean” refers to a numerical value that represents the central or standard value in a dataset, in particular the mode, median, or (most commonly) the mean, which is calculated by dividing the sum of the values ​​in the set by their number. “Mean” also refers to a single value (such as the mean, mode, or median) that summarizes or represents the general significance of an uneven set of values. 【0086】 As used herein, "non-natural" means anything that is manufactured, created, or constructed by humans, is artificial, or is an imitation of something that exists in nature. 【0087】 Routes of administration in various embodiments include, but are not limited to, topical, intraglandular, topical, transdermal, nasal, systemic administration (such as intravenous, intraarterial, intramuscular, subcutaneous, inhalation, rectal, cheek, vaginal, intraperitoneal, intra-articular, eye drops, ear, or oral administration) and eye drop administration (such as subconjunctival, topical, sub-Tenon, intravitreous, or anterior chamber administration) to the site requiring treatment (e.g., the surface of the eye in the treatment of dry eye, the knee in the treatment of osteoarthritis). As used herein, "systemic administration" refers to all non-cutaneous routes of administration, specifically excluding topical and transdermal routes of administration. Further methods for administering the MLC, PLC or their derivatives of this disclosure include intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intersternal injections and infusions. 【0088】 As used herein, the term “antagonist” refers to a substance that, when combined with a receptor, initiates a physiological response. Agonists activate cells by binding to their respective receptors or ligands on the cell. It can be any agent or entity capable of inhibiting the expression or activity of a protein, its polypeptide portion, or polynucleotide. Thus, an antagonist can act to prevent transcription, translation, post-transcriptional processing, or post-translational processing, or in other circumstances, to inhibit the activity of a protein, polypeptide, or polynucleotide in any case, either directly or indirectly. An antagonist can be, for example, a nucleic acid, a peptide, or any other suitable chemical compound or molecule, or any combination thereof. Furthermore, it will be understood that in indirectly impairing the activity of a protein, polypeptide, or polynucleotide, an antagonist may, in turn, affect the activity of cellular molecules that can act as regulators or the protein, polypeptide, or polynucleotide itself. Similarly, an antagonist may affect the activity of molecules that are themselves regulated or modulated by a protein, polypeptide, or polynucleotide. As used herein, the term "agonist activation" refers to the activation of a cell receptor or ligand induced by a receptor-specific agonist. 【0089】 "Donor platelets" refer to bone marrow-derived platelets physiologically produced within the body of a mammal (e.g., a human). Donor PRP refers to platelets prepared from the donor's blood. 【0090】 As used herein, the term “subject” refers to mammals, including but not limited to humans, pigs, horses, dogs or cats, or any other animals that may be susceptible to viral infection. The term “subject” encompasses a healthy population, a population potentially susceptible to a pathogen, or a patient suffering from a viral infection. 【0091】 The terms “drug,” “pharmaceutical,” or “compound” as used herein refer to a biological product or chemical entity, or a combination of a biological product and a chemical entity, administered to a human being in a therapeutic dose to treat, prevent, or control a disease or condition. A biological product or chemical entity may include, but is not limited to, antibodies or fragments thereof, low molecular weight compounds, and may also include, but is not limited to, larger compounds such as proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and their variants and combinations, nucleic acids, amino acids, or carbohydrate oligomers. 【0092】 The terms “drug,” “therapeutic composition,” or “therapeutic agent” can be used interchangeably and refer to a therapeutic agent. A drug may be selected from one or more of the following: proteins, peptides, aptamers, antibodies, or fragments thereof, chemical substances, small molecules, nucleic acid sequences, or nucleic acid analogs. A nucleic acid sequence may be RNA or DNA, may be single-stranded or double-stranded, and may be selected from nucleic acids, oligonucleotides, or nucleic acid analogs encoding a protein of interest, such as peptide nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA), etc. Examples of such nucleic acid sequences include, but are not limited to, RNAi, shRNAi, siRNA, microRNAi (mRNAi), antisense oligonucleotides, etc., but also include, but are not limited to, nucleic acid sequences encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, and small inhibitory nucleic acid sequences. Proteins and / or peptides or fragments thereof may be any protein of interest, such as, for example, mutant proteins; therapeutic proteins; or cleaved proteins in which the protein is not normally present in cells or is expressed at low levels. Proteins can also be selected from the group including mutant proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, midibodies, tribodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins, and fragments thereof. The drug can be conjugated into a culture medium where it comes into contact with cells and induces its effect. Alternatively, the drug may be intracellular, or intracellular, for the introduction of nucleic acid sequences into cells and their transcription, resulting in intracellular nucleic acid production and / or protein environmental stimulation. In some embodiments, the drug is any chemical substance, entity, or part, including but not limited to synthetic and naturally occurring non-proteinogenic entities. In some embodiments, the drug is a small molecule having a chemical moiety. For example, chemical moieties include unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties, including macrolides, leptomycin and related natural products or their analogues.The drug may be known to have the desired activity and / or properties, or it may be selected from a diverse library of compounds. 【0093】 As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. The term "antibody fragment" refers to a portion of an intact antibody, specifically the antigen-determining variable region of the intact antibody. 【0094】 The terms “culture conditions,” “culture medium,” or “culture medium” can be used interchangeably and refer to a medium for culturing cells that contains nutrients that maintain cell viability and support cell proliferation and maintenance or cell differentiation stages. In addition to the embodiments disclosed herein, cell culture media may contain any of the following in appropriate combination: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum substitutes, and other components such as peptide growth factors. Suitable cell culture media for different cell types are known to those skilled in the art. 【0095】 As used herein, terms such as “to treat,” “to treat,” and “treatment” refer to reducing or restoring a disorder and / or its associated symptoms. It will be understood that treating a disorder or condition does not require the elimination of the associated disorder, condition, or symptoms, although this is not excluded. 【0096】 As used herein, the term “PLC-enriched plasma” refers to enriched PRP or enriched plasma supplied by a combination of precursor cells for producing PLC or its derivatives, or other plasma sources such as donor-derived PRP, or lysates extracted from them. Where appropriate, the term “PLC-enriched plasma” may include growth factors, cytokines, or other agents from other sources that complement the therapeutic use of PLC or its derivatives. 【0097】 Where specifically stated or evident from the context, the term “or” is understood to be inclusive. Where not specifically stated or evident from the context, the terms “a,” “an,” and “the” are understood to be singular or plural. 【0098】 The word “substantially” does not exclude “completely”; for example, a composition that “substantially” does not contain Y may not contain Y at all. Where necessary, the word “substantially” may be omitted from the definitions in this disclosure. 【0099】 When used herein, the term “extracellular vesicles (EVs)” refers collectively to microvesicles and exosomes, which are generally very small (typically about 1 micron or less in diameter; microvesicles, typically about 200–1500 nm or less in diameter; exosomes, typically about 20–200 nm or less in diameter) phospholipid vesicles expelled from megakaryocytes or other cells. Extracellular vesicles (EVs) may contain or transport materials such as, but not limited to, nucleic acids (e.g., siRNA), growth factors, proteins, or exogenous genetic material (e.g., for gene therapy), and express extracellular markers of their parent cells. Megakaryocyte-derived extracellular vesicles (EVs) may play a role in several pathways, including hemostasis and inflammation, and in the treatment of various disorders, including, but not limited to, malignancies (e.g., neoplasms), Alzheimer's disease, and tumor progression and development. 【0100】 The term “cryopreservation medium” refers to a liquid medium (solution or suspension) capable of preserving the structure and metabolism of isolated cells against damage associated with a freezing event, either internal or external to the cell, and that is safe for injection into humans. The term further refers to a medium (solution or suspension) containing components including cryopreservatives that have been determined or are known to be safe for injection into humans. Preferably, the medium (solution or suspension) and the drugs, components, or elements of the medium, such as histidine (25-50 mM), are approved by U.S. regulatory agencies for injection into humans. “Cryoprotective agent” is a drug capable of conferring some degree of cryoprotection to cellular structure and metabolism upon freezing. Cryoprotective agents within the scope of this disclosure include arabinogalactan and its biological and functional equivalents, glycerol, propylene glycol, and albumin, such as human serum albumin, plasma, or serum. 【0101】 The term "therapy" is intended to encompass any form of treatment, prevention, or diagnosis, including treatment for both the cure and prevention of disease. Therefore, treatment of a healthy patient should be considered therapy. Therapy also encompasses symptom relief in addition to curative treatment of disease. 【0102】 The compositions and methods of this disclosure utilize compositions of megakaryocyte derivatives. These compositions comprise growth factors, cytokines, and other megakaryocyte-derived components of a protein or other configuration, and offer the unique advantage of maximizing therapeutic outcomes and minimizing side effects in the treatment, repair, or recovery of diseases, disorders, or injuries related to dry eye, osteoarthritis, tendon, ligament, bone repair, wound healing or wound healing-related disorders, alopecia, or skin rejuvenation or regeneration, for which adequate or consistent treatment is not available by conventional means. In some embodiments, the megakaryocyte derivative comprises megakaryocyte-like cells (MLCs) (including those of engineered MLCs). In some embodiments, the megakaryocyte derivative comprises MLC lysates. In some embodiments, the megakaryocyte derivative comprises PLCs or derivatives thereof (including those of engineered PLCs). 【0103】 In some embodiments, the megakaryocyte derivative is derived from the reprogramming of somatic cells, progenitor cells, or stem cells, and the product passes through a bioreactor. In some embodiments, the megakaryocyte derivative does not contain cancer cells. In some embodiments, the megakaryocyte derivative does not exhibit uncontrolled proliferation or tumorigenesis in vivo. 【0104】 In some embodiments, megakaryocyte derivatives restore goblet cell loss in dry eye disease. In some embodiments, megakaryocyte derivatives are used to treat or reverse symptoms of Sjögren's syndrome. In some embodiments, topical treatment of the target eye with a megakaryocyte derivative improves corneal barrier integrity compared to the target eye treated with a vehicle. In some embodiments, megakaryocyte derivatives contain higher concentrations of epitheliotropic factor and platelet-related factor compared to human blood-derived PRGF, TIMPS which inhibits MMP histolysis, interleukins or growth factors that polarize T cells and secrete dissipation-promoting cytokines, or a combination thereof. In some embodiments, megakaryocyte derivatives contain higher concentrations of epidermal growth factor (EGF) compared to human blood-derived PRGF. In some embodiments, megakaryocyte derivatives promote corneal epithelial cell proliferation. In some embodiments, growth factors can stimulate the migration and growth of corneal epithelial cells. In some embodiments, megakaryocyte derivatives contain immunomodulators that reduce immune cell-mediated cell loss. In some embodiments, megakaryocyte derivatives contain neurotrophic factors that protect corneal nerve cells and / or stimulate the regeneration of corneal nerve cells. In some embodiments, treatment with megakaryocyte derivatives helps preserve intact articular cartilage in subjects with osteoarthritis and / or improves / reverses the cartilage degeneration state compared to subjects treated with vehicle alone. In some embodiments, treatment with megakaryocyte derivatives reduces the rate of cartilage degeneration or reverses cartilage degeneration in subjects with osteoarthritis compared to subjects treated with vehicle alone. In some embodiments, treatment with megakaryocyte derivatives promotes wound healing in subjects treated with vehicle alone. In some embodiments, treatment with megakaryocyte derivatives promotes wound healing in diabetic subjects treated with vehicle alone. 【0105】 The compositions and methods of the present disclosure utilize the properties of PLC or its derivatives (including those of engineered PLC) to provide a unique opportunity to maximize therapeutic outcomes in the treatment, repair, or recovery of diseases, disorders, or injuries related to dry eye, osteoarthritis, tendons, ligaments, bone repair, wound healing or wound healing-related disorders, alopecia, or skin rejuvenation or regeneration, as well as to minimize side effects. 【0106】 Prior art protocols for platelet-rich plasma (PRP) concentration for use are essentially multi-step processes, which increases the risk of contamination and leads to product inconsistency and unreliability. For example, a typical PRP preparation involves multiple steps: (1) taking a small amount of venous blood (15-50 mL) from the patient's arm into an anticoagulant-containing tube; (2) the recommended temperature during processing is 21°C-24°C to prevent platelet activation during blood centrifugation; (3) centrifuging the blood at 1,200 rpm for 12 minutes; (4) separating the blood into three layers: an upper layer containing platelets and leukocytes, a thin middle layer (buffy coat) rich in leukocytes, and a lower layer containing erythrocytes; (5) transferring the upper and middle buffy layers into empty sterile tubes. The plasma is centrifuged again at 3,300 rpm for 7 minutes to help form a soft pellet (red blood cells and platelets) at the bottom of the tube; (6) The top two-thirds of the plasma is discarded as it is platelet-poor plasma; (7) The pellet is homogenized with the bottom one-third (5 mL) of the plasma to create injection-ready PRP. 【0107】 This disclosure eliminates many of the prior art steps involved in PRP and serum preparation, thereby minimizing the risk of contamination or impurities. Most importantly, this disclosure eliminates the need to collect venous blood, often a source of contamination (e.g., viruses such as HIV), unless it is definitively analyzed. In other words, this disclosure eliminates the need to collect blood or separate blood into different components, at least, before platelets are isolated. This is achieved by culturing a population of progenitor stem cells to a state in which they substantially differentiate into mature megakaryocytes. The mature megakaryocytes are then cultured in a bioreactor or fluid device, where the bioreactor gradient in the bioreactor mimics an endogenous platelet-producing environment to produce PLC (microcrystalline cell) or its derivatives. Subsequently, MLC, PLC, or their derivatives are collected from the bioreactor in quantities sufficient for use. In some embodiments, MLC, PLC, or their derivatives, or their lysates, can be used alone or in combination with EV. In some embodiments, MLC, PLC, or derivatives thereof, or their lysates, can be used to enrich donor platelets in platelet-rich plasma (i.e., PLC or genetically modified PLC is mixed with donor-based PRP). In some embodiments, MLC, PLC, or derivatives thereof can be used in combination with EVS or in combination with other agents disclosed herein. 【0108】 Megakaryocyte derivatives rich in growth factors and cytokines (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom), or precursor cells for producing megakaryocyte derivatives (MLC, PLC or their derivatives or their lysates, or PRP, multi-growth factor plasma (PRGF), or autologous serum (AS) derived therefrom), can be used in their intact form or in liquid or paste form, or can be mixed with other agents such as gels, ointments, creams, or other emulsifiers, acceptable diluents, carriers, or excipients, but not limited thereto. 【0109】 In some embodiments, the Disclosure further provides pharmaceutical compositions comprising megakaryocyte derivatives (e.g., MLC, PLC or derivatives thereof or lysates thereof, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing the megakaryocyte derivatives of the Disclosure (e.g., MLC or PLC or derivatives thereof, or lysates thereof, or PRP, PRGF, or AS derived therefrom), and (2) pharmaceutically acceptable expanders, carriers, or excipients. In some embodiments, the pharmaceutical composition comprises (1) a megakaryocyte derivative (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing a megakaryocyte derivative (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom), (2) a pharmaceutically acceptable expander, carrier, or excipient, and optionally (3) at least one further therapeutic agent. In some embodiments, the pharmaceutical composition comprises (1) a megakaryocyte derivative (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing a megakaryocyte derivative (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom), (2) a pharmaceutically acceptable expander, carrier, or excipient, and optionally (3) at least one further therapeutic agent and concentrate. 【0110】 Compositions containing megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) preferably include polysaccharides, for example, natural polysaccharides (such as hyaluronic acid, hydroxypropylcellulose, karya gum (KG), guar gum (GUG), or gellan gum (GEG)), skin fillers (e.g., Juvederm®, Juvederm® Ultra Plus, Perlane®, Belotero®, Restylane®), semi-synthetic or synthetic polysaccharides, synthetic polymers (e.g., poly(7-oxanorbornene-2-carboxylate), F127, or poly(lactic acid-coglycolic acid) (PLGA)), sodium citrate, calcium chloride, proteoglycans, adenine, guanine, cytosine, thiamine, precursor stem cells or their derivatives, vitamins, retinol, retinoic acid, and other compounds. Retinyl lumitate, acetate (e.g., tocopheryl acetate), phosphate (e.g., sodium ascorbyl phosphate), D-panthenol, peptides, recombinant growth factors, micronized human-identical hormones, amino acids, plant extracts, antioxidants, lipoic acid, DMAE, collagen, GAGs, trace elements, minerals, proteases, ceramides, polysaccharides, algae, marine extracts, monocytes, or any combination thereof may be concentrated with or formulated with. 【0111】 Further therapeutic agents include, but are not limited to, those described elsewhere in this disclosure. Compositions comprising megakaryocyte derivatives (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) may be administered topically (e.g., knee in osteoarthritis), parenterally, i.e., by injection, subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural, or topically, optically, or by inhalation, or rectally, vaginally, or sublingually. 【0112】 Therapeutic compositions and formulations thereof, comprising megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom), used in accordance with this disclosure, are prepared for storage by mixing the megakaryocyte derivative (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS) or precursor cells for producing megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or PRP, PRGF, or AS derived therefrom) having the desired purity with a pharmaceutically acceptable carrier, excipient, or stabilizer as needed (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or aqueous solution.Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers such as acetates, tris, phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol, etc.); low molecular weight (less than approximately 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins. Contains chlorine; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; isotonic agents such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose, or sorbitol; surfactants such as polysorbate; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).Other pharmaceutically acceptable carriers may include, but are not limited to, binders (e.g., pre-gelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, calcium hydrogen phosphate, etc.), lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), disintegrants (e.g., starch, sodium starch glycolate, etc.), or wetting agents (e.g., sodium lauryl sulfate, etc.), water, salt solutions, alcohols, polyethylene glycol, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.). Pharmaceutical formulations intended for in vivo administration are generally sterile. This is easily achieved by filtration through a sterile filtration membrane. 【0113】 Formulations comprising the compositions described herein include granules, tablets, suspensions in liquid carriers, capsules, or powders. The formulations described herein may also contain, as necessary, more than one active compound with respect to the specific indication being treated, preferably having complementary activities that do not adversely affect each other. For example, formulations comprising the compositions described herein, in addition to megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or PRP, PRGF, or AS derived therefrom), may preferably contain further injury-healing agents, such as anti-inflammatory agents or opioid drugs in a single formulation. Alternatively, or further, the composition may further contain cytotoxic agents, cytokines, growth inhibitors, antihormonal agents, and / or cardioprotective agents. Such molecules are appropriately present in combination in amounts effective for the intended purpose. 【0114】 The active ingredient (i.e., MLC or PLC or their derivatives or their solutions, or PRP, PRGF, or AS derived therefrom) may also be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980). 【0115】 The active ingredient (i.e., MLC, PLC or their derivatives or lysates, or PRP, PRGF, or AS derived therefrom) may also be delivered in a bioactive scaffold made from natural (e.g., protein-based) or synthetic (polymer or ceramic-based) biomaterials. In addition to megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or lysates, or PRP, PRGF, or AS derived therefrom), the bioactive scaffold may optionally contain growth factors and other bioactive molecules. These include epidermal growth factors. Some examples include TGF-alpha, TGF-beta, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, insulin-like growth factor, keratinocyte growth factor, and osteomorphoproteins. Growth factors may also be introduced into the scaffold before or after the introduction of megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or lysates, or PRP, PRGF, or AS derived therefrom). 【0116】 A sustained-release preparation may be prepared. A suitable example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing a megakaryocyte derivative (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing a megakaryocyte derivative (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom), the matrix being in the form of a molded article, such as a film, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), L-glutamic acid and gamma-ethyl-L-glutamate copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuproid acetate), and poly-D-(-)-3-hydroxybutyric acid. Examples of biocompatible materials that may be present in hydrogels include permeable compositions or forms such as polyvinyl alcohol, polyvinylpyrrolidone and polyacrylamide, polyethylene oxide, and poly(2-hydroxyethyl methacrylate); natural polymers such as polysaccharides, gums, and starches; and poly[alpha(4-aminobutyl)]-1-glycolic acid, polyethylene oxide, polyorthoesters, silk elastin-like polymers, alginates, EVAc (poly(ethylene-co-vinyl acetate)), and poly Examples of soybean matrices include (D,L-lactide-co-glycolide) copolymers and microspheres such as poly(L-lactide), poly(N-isopropylacrylamide)-b-poly(D,L-lactide), crosslinked with glyoxal, and reinforced with bioactive fillers, such as hydroxyl apatite, poly(epsilon-caprolactone)-poly(ethylene glycol) copolymer, poly(acryloylhydroxyethyl) starch, polylysine-polyethylene glycol, or agarose.In one embodiment, the hydrogel comprises poloxamer, polyacrylamide, poly(2-hydroxyethyl methacrylate), carboxyvinyl polymer (e.g., Carbopol 934, Goodrich Chemical Co.), cellulose derivatives such as methylcellulose, cellulose acetate, and hydroxypropylcellulose, polyvinylpyrrolidone, or polyvinyl alcohol, or a combination thereof. In some embodiments, the hydrogel comprises collagen such as hydroxylated collagen, fibrin, polylactic acid-polyglycolic acid, or polyanhydride. Other examples include, but are not limited to, any biocompatible polymers that are hydrophilic, hydrophobic, or amphiphilic, such as ethylene vinyl acetate copolymer (EVA), polymethyl methacrylate, polyamide, polycarbonate, polyester, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, N-isopropylacrylamide copolymer, poly(ethylene oxide poly(propylene oxide)) block copolymer, poly(ethylene glycol) / poly(D,L-lactide-co-glycolide) block copolymer, polyglycolide, polylactide (PLLA or PDLA), poly(caprolactone) (PCL), or poly(dioxanone) (PPS). The following polymers may be used, for example, natural polymers such as alginate, agarose, starch, fibrin, collagen, gelatin, chitin, glycosaminoglycans, for example, hyaluronic acid, dermatan sulfate and chrondrotin sulfate, as well as microbial polyesters, for example, hydroxyalkanoates such as hydroxyvalerate and hydroxybutyrate copolymers, as well as synthetic polymers, for example, poly(orthoesters) and polyacid anhydrides, and homo and copolymers of glycosides and lactides (for example, poly(L-lactide), poly(L-lactide-co-D,L-lactide), poly(L-lactide-co-glycolide), polyglycolide and poly(D,L-lactide), poly(D,L-lactide-coglycolide), poly(lactic acid coglycine) and polycaprolactone).Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow for molecular release over a period of 100 days, while certain hydrogels release proteins in a shorter timeframe. Encapsulated megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom) may denature or aggregate as a result of exposure to water at 37°C if they remain in the body for extended periods, leading to loss of biological activity and possible changes in activity. 【0117】 Megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) can be formulated in any suitable form for delivery to target cells / tissues. For example, megakaryocyte derivatives (e.g., MLC or MLC derivatives or PLC or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or PRP, PRGF, or AS derived therefrom) can be formulated as immunoliposomes. "Liposomes" are vesicles composed of various types of lipids, phospholipids, and / or surfactants that are useful for the delivery of drugs to mammals. The components of liposomes are generally arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or lysates thereof, or PRP, PRGF, or AS derived therefrom) are prepared by methods known in the art, as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); US Pat. Nos. 4,485,045 and 4,544,545; and International Publication No. 97 / 38731, published on October 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. 【0118】 Useful liposomes can be produced by reverse-phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a specified pore size to obtain liposomes of the desired diameter. Another therapeutic agent may be contained within the liposomes as needed. See Gabizon et al., J. National Cancer Inst. 81(19):1484 (1989). 【0119】 Preparations intended for in vivo administration must be sterile. This can be easily achieved by filtration using a sterile filtration membrane. 【0120】 To treat injuries (e.g., osteoarthritis, regenerating bone tissue to influence the processes of bone regeneration and repair), or to regenerate aging skin or hair, in one embodiment, megakaryocyte derivatives (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) are administered via any of the routes disclosed herein. The dose administered via any of these routes is approximately 0.1 micrograms / m² per single dose. 2 ~Approximately 10,000 micrograms / m² 2 The dosage range is generally once a day or once a week, totaling one, two, three, or four doses, or multiple doses as needed. Alternatively, the dosage range is approximately 0.1 micrograms / m². 2 ~Approximately 1,000 micrograms / m² 2 Approximately 0.1 micrograms / m² 2 ~Approximately 800 micrograms / m² 2 Approximately 0.1 micrograms / m² 2 ~Approximately 600 micrograms / m² 2 Approximately 0.1 micrograms / m² 2 ~Approximately 400 micrograms / m²2 Approximately 0.1 micrograms / m² 2 ~Approximately 500 micrograms / m² 2 Approximately 0.1 micrograms / m² 2 ~Approximately 300 micrograms / m² 2 Approximately 0.1 micrograms / m² 2 ~Approximately 200 micrograms / m² 2 , and approximately 0.1 micrograms / m² 2 ~Approximately 200 micrograms / m² 2 The dosage may be once daily, once weekly, multiple times weekly, less than once daily or more than once daily, two to three times daily, multiple times monthly, once daily, once weekly or once monthly, or intermittently on a daily, weekly or monthly basis (for example, one or more doses administered daily, weekly, every other week, every three weeks or monthly) to reduce or alleviate symptoms of damaged or aging skin or diseases associated with damaged or aging skin. Administration may be continued at any of the disclosed intervals until remission of the damaged or aging skin or treatment of symptoms associated with the disease is achieved. Administration may be continued after remission or alleviation of symptoms is achieved if such remission or alleviation is prolonged by continued administration. 【0121】 The emulsifiers may be natural or synthetic and include, but are not limited to, cationic, e.g., benzalkonium chloride, benzethonium chloride; anionic, e.g., alkaline soaps (sodium or potassium oleate); amine soaps (triethanolamine stearate); detergents (sodium lauryl sulfate, sodium dioctyl sulfosuccinate, sodium doxate); and nonionic, e.g., sorbitan esters (Span®), polyoxyethylene derivatives of sorbitan esters (Tween®), or glyceryl esters. 【0122】 Other agents that may be used as emulsifiers include deoxycholic acid, diacetyl tartrate, egg yolk, glycerol, gum, Irish moss (carrageenan), lecithin, mono and diglycerides, monosodium phosphate, monostearate, bovine bile extract, propylene glycol, soap, and taurocholic acid (or its sodium salt). 【0123】 In some embodiments of this disclosure, megakaryocyte derivatives (e.g., MLC, PLC or derivatives thereof or lysates thereof, or PRP, PRGF, or AS derived therefrom), or precursor cells for producing megakaryocyte derivatives (e.g., MLC or PLC or derivatives thereof, or PRP, PRGF, or AS derived therefrom), are plasmalights. Plasmalights are a group of well-balanced crystalloid solutions. Plasmalights closely mimic human plasma in terms of their electrolytes, osmotic pressure, and pH. These solutions also have further buffering capacity and contain anions such as acetates, glucons, and even lactates, which are converted to bicarbonates, CO2, and water. 【0124】 In some embodiments, MK and platelets are also human induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), immortalized megakaryocyte progenitor cells, and CD34, as disclosed in the concurrently pending U.S. Patent Application No. 16 / 975,918 filed June 25, 2020 and the PCT application Provisional Application No. 63 / 025,209 filed May 15, 2020, which are incorporated herein by reference in their entirety. + Umbilical cord blood stem cells (UCB cells) (e.g., human CD34) + Umbilical cord blood stem cells, CD34 + Mobilized peripheral blood cells (MPB cells) (e.g., CD34) + Human mobilized peripheral blood, or CD34 +Hematopoietic stem cells may be derived from bone marrow cells, but are not limited to these. UCB cells are pluripotent stem cells derived from blood remaining in the placenta and attached umbilical cord after birth. MPB cells are pluripotent stem cells derived from volunteers whose stem cells are mobilized into the bloodstream by administration of G-CSF or similar drugs. 【0125】 In some embodiments, MK and platelets may be derived from other stem cell types, including but not limited to mesenchymal stem cells (MSCs) (e.g., adipose-derived mesenchymal stem cells (AdMSCs)) or mesenchymal stems from other sources. 【0126】 AdMSCs originate from white adipose tissue, specifically the mesoderm during embryonic development, and are present in all mammalian species, located throughout the body. Due to their wide availability and ability to differentiate into other mesodermal tissue types, including bone, cartilage, muscle, and fat, ASCs can serve a variety of purposes. 【0127】 In this disclosure, the stem cell culture medium may be maintained independently of germinal fibroblast feeder cells and / or animal serum. In some embodiments, serum-free feeder cell-free alternatives may be used in this method. Extracellular vesicles (EVs) 【0128】 In some embodiments, the Disclosure comprises microvesicles and exosomes (collectively referred to as extracellular vesicles (EVs)) or derivatives thereof, which are produced as MLCs or PLCs or their derivatives or mixtures thereof lysates. Given that EVs or their derivatives carry growth factors, receptors, bioactive lipids, nucleic acids such as mRNA and microRNA (miRNA) or siRNA, and proteins, they are capable of delivering important payloads to recipient cells (e.g., osteoarthritis of the knee or skin or damaged organ or tissue for use in regenerative medicine), for example, to treat or restore diseases, disorders, or injuries associated with dry eye, osteoarthritis, tendons, ligaments, bone repair, wound healing or wound healing-related disorders, alopecia, or in skin rejuvenation or regeneration, supplementing MLCs or PLCs or their derivatives or lysates with further growth factors or other molecules would provide an even richer resource. 【0129】 The extracellular vesicles (EVs) or their derivatives may be isolated and purified, essentially separated from mixtures containing the MLCs or PLCs or their lysates. Due to their ability to travel extensively throughout the body, the isolated or purified extracellular vesicles (EVs) or their derivatives may exert significant therapeutic effects when administered to a patient on their own. EVs have fundamental immunomodulatory potential for treating or inhibiting diseases or disorders or injuries associated with dry eye, osteoarthritis, tendons, ligaments, bone repair, wound healing or wound healing-related disorders, alopecia, or skin rejuvenation or regeneration. EVs may also be used as drug delivery systems, as they are capable of crossing biological barriers such as the blood-brain barrier and synovial membrane. 【0130】 Preferably, the EV or its derivatives may be internally translocated by the recipient cell after receptor-ligand interaction, and a variety of bioactive molecules of cell origin, such as proteins, bioactive lipids, and nucleic acids, may be translocated along with proteins expressed on the EV surface. 【0131】 In some embodiments, extracellular viable cells (EVs) or their derivatives may directly activate recipient cells (e.g., donor platelets) by acting as signaling complexes. For example, EVs or their derivatives may bind to platelets via P-selectin glycoprotein ligand-1 expressed on their surface, and EVs or their derivatives from neutrophils expressing Mac-1 may induce activation of donor platelets in patients in need. Such activation is preferable because it promotes the activation of exogenous mechanisms that can enhance treatments for or recovery from diseases, disorders, or injuries associated with dry eye, osteoarthritis, tendons, ligaments, bone repair, wound healing or wound healing-related disorders, alopecia, or skin rejuvenation or regeneration. 【0132】 Compositions and methods comprising the extracellular vesicles (EVs) or derivatives thereof of this disclosure can be used, for example, with vectors, such as adenoviruses and lentiviruses, to obtain novel microvesicles or exosome gene (e.g., for gene therapy), peptides (for growth factors), or nucleic acids (e.g., siRNA or microRNA) delivery vehicles for use in skin rejuvenation or regeneration, or in the delivery of genes, proteins, peptides, or nucleic acids for use in cell or gene therapy. Packaging within extracellular vesicles (EVs) offers several advantages, such as protecting molecules from harmful cellular events that could neutralize the naked gene. Manipulated extracellular vesicles (EVs) can be used to deliver drugs to specific sites of tissue damage, including but not limited to dry eye, osteoarthritis, tendons, ligaments, bone repair, wound healing or wound healing-related disorders, and alopecia, or in skin rejuvenation or regeneration. 【0133】 In some embodiments, these isolated extracellular vesicle (EV) derivatives can be stored until use by freezing at ultracold temperatures, for example, -80°C, in the presence of cryopreservatives such as dimethyl sulfoxide (DMSO) and glycerol, which are subsequently used at optimal concentrations. 【0134】 In some embodiments, the average diameter of extracellular vesicles (EVs) derived from a population of iPSC-derived platelets is less than 50% of the diameter of extracellular vesicles (EVs) derived from a population of donor-derived platelets having approximately the same number of platelets as the iPSC-derived platelets. In some embodiments, megakaryocytes or platelets are genetically modified to contain nucleic acid molecules encoding a therapeutic agent. 【0135】 Extracellular vesicles (EVs) are intracellular-sized particles consisting of a membrane lipid bilayer and cellular contents. Extracellular vesicles (EVs) isolated or purified from mixtures containing MLCs, PLCs, their lysates, or combinations thereof may exert both anti-inflammatory and pro-inflammatory functions and have potential as vehicles for drug delivery. 【0136】 In some embodiments, the diameters of the extracellular vesicles (EVs) are 0.1 and 4 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) are 0.1 and 3 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) are 0.1 and 2.5 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) are 0.1 and 2 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) are 0.1 and 1.5 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) are 0.1 and 1.0 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) are 0.1 and 0.9 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) are 0.1 and 0.8 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) are 0.1 and 0.7 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.1 and 0.6 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.1 and 0.5 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.1 and 0.4 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.1 and 0.3 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.1 and 0.2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.2 and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.3 and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.4 and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.5 and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.6 and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.7 and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.8 and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.9 and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.2 and 2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.3 and 2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.4 and 2 μm.In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.5 and 2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.6 and 2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.7 and 2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.8 and 2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 0.9 and 2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 1.0 and 2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 1.5 and 2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) is 2.0 and 2.5 μm. 【0137】 The extracellular vesicle (EV) derivative can be conjugated to one or more cytotoxic agents by the mechanism disclosed above. The one or more cytotoxic agents can also be absorbed by the extracellular vesicle (EV) derivative by the mechanism disclosed above. The cytotoxic agents are also disclosed above. Diseases and disorders that can be cured or mitigated by the use of the EV derivative alone or in combination with the MLC, PLC, their lysates or derivatives disclosed herein are also disclosed below. 【0138】 In some embodiments, EVs may be developed for therapeutic use independently of MLCs, PLCs, their lysates, or their derivatives, whether modified (e.g., bioengineered or conjugated). For example, patients requiring treatments primarily involving microvesicles or their derivatives may receive microvesicle-based treatments, exosome-based treatments, or a combination of both. For instance, MVs or exosomes incorporating exogenous growth factors, cytokines, or siRNAs may be used for efficient silencing of targeted MAPK genes in monocytes and lymphocytes, or to target bone tissue, for example, by delivering regenerative growth factor siRNAs (e.g., VEGF-siRNA) to influence bone regeneration and repair processes as well as alopecia processes. Preferably, MVs may be used as more efficient delivery vehicles to deliver specific targets of novel therapeutics without immunogenicity and side effects. 【0139】 In some embodiments, the EV-based treatment may be administered before the treatment with a megakaryocyte derivative (e.g., MLC, MLC lysate, PLC, or PLC lysate). In some embodiments, the treatment with a megakaryocyte derivative (e.g., MLC, MLC lysate, PLC, or PLC lysate) may be administered before the EV-based treatment. In some embodiments, the megakaryocyte derivative (e.g., MLC, PLC, their lysates, or combinations thereof) and EV are administered as a mixture. It is also conceivable that a mixture containing a megakaryocyte derivative (e.g., MLC, PLC, their lysates, or combinations thereof) and EV is administered, followed by a treatment regimen that, depending on the patient's needs, essentially contains EV or its derivatives, or is essentially a treatment based on a megakaryocyte derivative (e.g., MLC or PLC or their derivatives, or their lysates). 【0140】 Some embodiments of this disclosure provide compositions and cryopreservatives suitable for cryopreservation, comprising megakaryocyte derivatives (e.g., MLC, PLC or derivatives thereof, or lysates thereof, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC or PLC or derivatives thereof, or PRP, PRGF, or AS derived therefrom). In some embodiments, the cryopreservative is a sugar, alcohol, polymer, protein, or a combination thereof. In some embodiments, the cryopreservative is DMSO, glycerol, trehalose, cellulose, or a combination thereof. In some embodiments, the cryopreservative is DMSO. In some embodiments, the cryopreservative is glycerol or arabinogalactan. In some embodiments, the cryopreservative is DMSO and glycerol. In some embodiments, the cryopreservative is trehalose or propylene glycol or albumin or a combination thereof. In some embodiments, the cryopreservative is cellulose. Combinations comprising one or more cryopreservatives are also included in this disclosure. In some embodiments, the composition is frozen, i.e., stored at a temperature of approximately -80°C to -200°C. Cryoprotective agents can be classified into permeable and non-permeable types. Non-permeable cryoprotective agents alter only the freezing properties of the extracellular medium, while permeable cryoprotective agents can alter both the intracellular and extracellular medium composition. 【0141】 Megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or PRP, PRGF, or AS derived therefrom) are, in non-limiting embodiments, rich in growth factors or angiopoietin-1, bFGF, EGF, FGF (e.g., FGF-2), HGF, IGF-I, IGF-II, PDAF, PDEGF, PDGF (e.g., PDGF-AA, PDGF-BB, PDGF-AA / BB), TGF-beta (e.g., TGF-β1, TGF-β2, and TGF-β3), VEGF (e.g., VEGF-A, VEGF-C), IFN-γ, site These may include, but are not limited to, growth factors such as kines (e.g., IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-12p40, IL-12p70, IL-13, IL-17A, IL-23, TNF-A, MIG, MCP-1, IP-10), chemokines (e.g., ENA-78 (CXCL5), IL-8 (CXCL8)), monocyte chemotactic proteins (e.g., MCP-3 (CCL7), MIP-1A (CCL3), NAP-2 (CXCL7), PF4 (CXCL4)), or inflammatory mediators (e.g., PGE2), macrophage inflammatory protein-1 (MIP-1), or activated regulated normal T cell expression secretion factor (RANTES), and may be supplemented with these factors, the concentrations of which are often higher than those obtained from donor platelets. MLC, PLC or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom, or MLC, PLC or their derivatives (e.g., lysates from MLC, PLC or derivatives thereof), or concentrates of growth factors or cytokines in PRP, PRGF, or AS concentrated from precursor cells for producing MLC, PLC or their derivatives, or PRP, PRGF, or AS derived therefrom, may be diluted as desired as required by the subject.In some embodiments, MLCs and PLCs may be genetically engineered to express one or more of the growth factors discussed herein. Non-limiting examples of endogenous PLC-based growth factors are shown in Figures 1A–1C (n=8 each). Figure 1A provides a profile of growth factors and cytokines in PLCs compared to donor PRP and washed donor platelets. Figure 1B provides the most different factors among PLCs compared to donor PRP and washed donor platelets. Figure 1C provides a summary of pro-inflammatory / anti-inflammatory markers associated with dry eye disease. 【0142】 Megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC or their derivatives, or PRP, PRGF, or AS derived therefrom) or compositions thereof are used in sprained ligaments (connecting bone to bone), bruised or lacerated muscles, ruptured tendons, osteoarthritis, fractured bones that may crack or break, or surrounding tissues. It can be administered or given to damaged tissues of the musculoskeletal system, such as injured tissues, bone dislocations (i.e., when the bones of a joint can be completely separated from each other (called dislocation) or simply partially displaced (called subluxation)), sprains, contusions, or other musculoskeletal injuries, completely or partially torn tendons, muscle spasms, neuralgia, whiplash, sports injuries, myocardial infarction or ischemia-related disorders (e.g., limb ischemia, lower limb ischemia, myocardial ischemia, organ ischemia, ischemic heart disease, etc.), surgical procedures that are difficult to heal, lung diseases, heart diseases, and other damaged tissues of the musculoskeletal system. 【0143】 The site of tissue damage or injury can be determined by well-established techniques, including but not limited to imaging studies such as MRI, X-rays, CT scans, and X-rays that may be performed to confirm any fractures and dislocations that may be present. Furthermore, X-rays may show abnormal bone positioning that may indicate sprains or other soft tissue injuries. Magnetic resonance imaging (MRI) can reveal soft tissues that are not normally visible on X-rays. Therefore, MRI is useful for detecting injuries to tendons, ligaments, cartilage, and muscles. Other techniques, including but not limited to positron emission tomography (PET), single-photon emission computed tomography (SPECT), electrical impedance imaging (EIT), electrical source imaging (ESI), magnetic source imaging (MSI), laser optical imaging (NOGA mapping), and ultrasound techniques, may be applied to determine the site of injury. A physician may consult with the patient before one or more of the above tests are performed. 【0144】 Megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or lysates thereof, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or PRP, PRGF, or AS derived therefrom, or compositions thereof) can be administered via various routes, including but not limited to systemic and local administration, using implantable devices such as stents, meshes (e.g., polymer meshes or bioabsorbable meshes), adhesive biomaterials (e.g., naturally derived or synthetic biopolymers), or other devices known to those skilled in the art. For example, variables such as appropriate timing, administration frequency, location, and technique for knee injections (e.g., intravenous or intradermal injections) in patients with osteoarthritis of the knee vary depending on the application of one or more megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or lysates thereof). Megakaryocyte derivatives (MLC or PLC or derivatives thereof or lysates thereof) may be administered daily, weekly (PLC injection every three weeks), or monthly (two injections per month, or three injections at 15-day or 21-day intervals; the treatment regimen may vary depending on the patient's needs). The location and technique for administering megakaryocyte derivatives (e.g., MLC, PLC or derivatives thereof or lysates thereof, or PRP, PRGF, or AS derived therefrom) (e.g., local injection into the knee) may be lateral, superlateral, parapatellar, and lateral mediapatellar, among others. In each such treatment, one or more other therapeutic agents may be co-administered simultaneously or at cyclical intervals from each other, as needed. 【0145】 When stents are used, they may be closed-cell or open-cell stents, both of which are well known to those skilled in the art. Megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) may be directly bound to the metal stent or to a matrix polymer that acts as a drug reservoir to ensure source retention and uniform distribution on the stent during deployment. The type, composition, and design of the polymer coated onto the stent generally determine the elution kinetics of the megakaryocyte derivative (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) over a period of several weeks to several months after in-situ implantation. Coating materials can be classified as organic versus inorganic, bioerodable versus non-biodegradable, and synthetic versus naturally occurring. 【0146】 In some embodiments, a composition comprising megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom) comprises 0.01 to 1% by weight, 1 to 25% by weight, 25 to 50% by weight, 50 to 75% by weight, or 75 to 100% by weight of megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or their lysates) or platelet-rich plasma (PRP), PRGF, plasma, serum, or other blood derivatives derived therefrom. 【0147】 In some embodiments, the composition comprises 1-25% by weight, 25-50% by weight, 50-75% by weight, and 75-98% by weight of an expander. In some embodiments, the composition comprises 1-25% by weight, 25-50% by weight, 50-75% by weight, and 75-98% by weight of at least one excipient or carrier. The weight percentages of megakaryocyte derivatives (e.g., MLC or PLC or their derivatives or lysates) or platelet-rich plasma (PRP), PRGF, plasma, serum, or other blood derivatives derived therefrom, expanders, excipients, or carriers can be adjusted as appropriate. For example, in one aspect of the present disclosure, the composition may comprise a) 1 to 50% by weight of a megakaryocyte derivative (e.g., MLC or PLC or derivatives thereof or lysates thereof) or platelet-rich plasma (PRP), PRGF, plasma, serum, or other blood derivatives derived therefrom; b) 1 to 25% by weight of a volume extender; and / or c) 50 to 98% by weight of at least one excipient or carrier. 【0148】 In some embodiments, the adhesive biopolymer material may include, but is not limited to, polycarbophil (PCP), xanthan gum, pectin, hydroxypropyl methylcellulose (HPMC) or hypromellose, Carbopol 1342P, Carbopol 974P, chitosan, Carbopol 971P, hydroxypropyl methylcellulose (Methacel K100M), CMC-Na, hydroxypropyl methylcellulose (Methacel K15M), gelatin, acacia gum, or combinations thereof. When MLC, PLC, or derivatives thereof or their lysates, or PRP, PRGF, or AS derived therefrom are contained in the adhesive biomaterial, the adhesive bioagent adheres to the target site to extend the retention time of MLC, PLC, or derivatives thereof or their lysates, or PRP, PRGF, or AS derived therefrom in lesions (e.g., ocular lesions), and to improve the therapeutic effect of local diseases (e.g., dry eye diseases). Higher local drug concentrations and close contact with the absorption site can not only promote drug absorption but also increase the concentration gradient. Adhesive biopolymers can modulate transport pathways by opening epithelial tight junctions, thereby facilitating the diffusion of megakaryocyte derivatives (e.g., MLC, PLC, or their derivatives or lysates thereof, or PRP, PRGF, or AS derived therefrom). Furthermore, megakaryocyte derivatives (e.g., MLC, PLC, or their derivatives or lysates thereof, or PRP, PRGF, or AS derived therefrom) may adhere directly to the mucosa and be absorbed by mucosal capillaries, increasing bioavailability. In addition, the use of adhesive biomaterials enables the preparation of controlled-release formulations of megakaryocyte derivatives (e.g., MLC, PLC, or their derivatives or lysates thereof, or PRP, PRGF, or AS derived therefrom), thereby reducing the frequency of administration to improve the health of patients requiring therapy with megakaryocyte derivatives (e.g., MLC, PLC, their derivatives, and / or lysates therefrom). 【0149】 In some embodiments, megakaryocyte derivatives (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) may be conferred to dry eye disease. Dry eye disease is classified into two categories: (i) tear-deficient and (ii) evaporative. Sjögren's syndrome or non-Sjögren's syndrome encompasses exocrine disorders in which tear-deficient secretion occurs due to autoimmune processes affecting the lacrimal glands, salivary glands, and other organs of the body (Sjögren's syndrome), or non-Sjögren's syndrome caused by lacrimal gland disorders or lacrimal gland obstruction and reflex changes without the involvement of autoimmune factors. Some causes of Sjögren's syndrome or non-Sjögren's syndrome include age-related dry eye, congenital analcyemia, familial autonomic dysfunction, sarcoidosis, lymphoma, AIDS (acquired immunodeficiency syndrome), denervation, lacrimal gland obstruction as seen in pemphigus, trigeminal nerve injury, diabetes, neuropathy, contact lens use, and motor reflex block resulting from VII-related injury. Evaporative causes of dry eye disease include oil deficiency, eyelid changes, contact lens use, or ocular surface diseases such as allergic conjunctivitis, and some iatrogenic dry eye that occurs after the use of systemic or topical drug therapy, or after surgery or non-surgical procedures. 【0150】 Megakaryocyte derivatives (e.g., MLC, PLC, or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC, or their derivatives, or PRP, PRGF, or AS derived therefrom) may be imparted to eye drops. For example, 2-3 milliliters of this concentrate may be placed in sterile eye drops. The eye drops may be maintained at -20°C for long-term storage. If the use of eye drops containing megakaryocyte derivatives (e.g., MLC, PLC, or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) is desired, the patient may thaw the eye drops and maintain them at +4°C. The patient may use these eye drops 1-6 times a day for 1 month, 2 months, 3 months, or longer. In some embodiments, the patient may use the eye drops once a day for 1 week, 2 weeks, 3 weeks, or 4 weeks, or longer. 【0151】 In some embodiments, megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or PRP, PRGF, or AS derived therefrom) may be administered via injection or topically to areas of skin such as aging skin on the face, scalp, neck, chest, hands, arms, legs, abdomen, or buttocks. It is assumed that megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or PRP, PRGF, or AS derived therefrom) may be administered to the scalp to extend the lifespan of hair follicles and increase hair growth. For use on the skin or scalp, megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC or their lysates, or their derivatives, or PRP, PRGF, or AS derived therefrom) are provided in a composition comprising a cosmetically acceptable carrier. For example, one aspect of the present disclosure provides a method for generating hair follicles in a target scalp or area of ​​hair loss, comprising contacting the scalp or area of ​​hair loss with a megakaryocyte derivative (e.g., MLC, PLC or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or PRP, PRGF, or AS derived therefrom).Other precursor cells capable of differentiating into hair follicle cells may also be included, such that the precursor cells are inducible cells capable of inducing the differentiation of uninvolved epidermal cells into hair follicle cells when combined with the megakaryocyte derivatives of this disclosure (e.g., MLC, PLC or their derivatives or lysates thereof, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC, their lysates, or their derivatives). In some embodiments, megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC or their lysates, or their derivatives, or PRP, PRGF, or AS derived therefrom) (i.e., a source for PRP, PRGF, or AS) are conferred first, followed by inducible cells; or PRP, PRGF, or AS or their sources and inducible cells are conferred simultaneously; or inducible cells are conferred first, followed by PRP, PRGF, or AS or a source for PRP, PRGF, or AS. Except in cases of simultaneous conferral, one component may be conferred within hours, days, or weeks of the other, depending on the requirements for the desired conferral of the subject. 【0152】 In some embodiments, the megakaryocyte derivatives (e.g., MLC, PLC, their derivatives, or their lysates) are preferably matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 3 (MMP-3), matrix metalloproteinase 7 (MMP-7), matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 10 (MMP-10), matrix metalloproteinase 12 (MMP-12), matrix metalloproteinase -ase 13 (MMP-13), Tissue Matrix Metalloproteinase Inhibitor 1 (TIMP-1), Tissue Matrix Metalloproteinase Inhibitor 2 (TIMP-2), Tissue Matrix Metalloproteinase Inhibitor 3 (TIMP-3), Tissue Matrix Metalloproteinase Inhibitor 4 (TIMP-4), Transforming Growth Factor Beta-1 (TGFβ1), Transforming Growth Factor Beta-2 (TGFβ2), Transforming Growth Factor Beta-3 (TGFβ3), Angiopoietin-2 (Ang-2), Osteogenesis Impermeable Protein 9 (BMP-9), epidermal growth factor (EGF), endoglin, endothelin-1, fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2), follistatin, granulocyte colony-stimulating factor (G-CSF), heparin-bound EGF-like growth factor (HB-EGF), hepatocyte growth factor (HGF), interleukin-8 (IL-8), leptin, placental growth factor (PLGF), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), soluble CD40L (sCD40L), eotacin Syn, Fms-like tyrosine kinase receptor 3 ligand (FLT-3L), fractalkine, growth-regulating oncogene α (GROα), interferon alpha 2 (IFN-α2), interferon gamma (IFN-γ), interleukin 1 alpha (IL-1α), interleukin 1 beta (IL-1β), interleukin 1 receptor antagonist (IL-1RA), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6),Interleukin 7 (IL-7), Interleukin 8 (IL-8), Interleukin 9 (IL-9), Interleukin 10 (IL-10), Interleukin 12p40 (IL-12p40), Interleukin 12p70 (IL-12p70), Interleukin 13 (IL-13), Interleukin 15 (IL-15), Interleukin 17A (IL-17A), Interleukin 17E (IL-17E (or IL-25)), Interleukin 17F (IL-17F), Interleukin 18 (IL-18), Interleukin 22 (IL-22), Interleukin 27 (IL-27), Interferon-gamma-inducible protein 10 (IP-10), Monocyte chemotactic protein-1 (MCP-1), Monocyte chemotactic protein-3 (MCP-3), Macrophage-derived proteins The biomarker comprises one or more biomarkers selected from mokines (MDC), gamma-induced monokines (MIG (or CXCL9)), macrophage inflammatory protein-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB (PDGF-AB), activated regulated normal T cell expression secretion factor (RANTES), transforming growth factor alpha (TGFα), tumor necrosis factor α (TNFα), tumor necrosis factor β (TNFβ), vascular endothelial growth factor A1 (VEGF-A1), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), angiopoietin-1, growth-inducing ligand (APRIL), and B cell activator (BAFF). In some embodiments, megakaryocyte derivatives (e.g., MLC, PLC, their derivatives or lysates) are preferably fibroblast growth factor-2 (FGF-2); hepatocyte growth factor (HGF); insulin-like growth factor 1 (IGF-1),The biomarker comprises one or more biomarkers selected from the following: activated regulated normal T cell expression secretion factor (RANTES); neuronal growth factor (NGF); vascular endothelial growth factor (VEGF-A); vascular endothelial growth factor (VEGF-C); epidermal growth factor (EGF); transforming growth factor-β1 (TGF-β1); transforming growth factor-β2 (TGF-β1); platelet-derived growth factor-AA (PDGF-AA); platelet-derived growth factor-BB (PDGF-BB); platelet-derived growth factor-AA / BB (PDGF-AA / BB); interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-12p40 (IL-12p40); interleukin-12p70 (IL-12p70); tissue metalloproteinase inhibitor 1 (TIMP-1); tissue metalloproteinase inhibitor 2 (TIMP-2); and tissue metalloproteinase inhibitor 3 (TIMP-3). In some embodiments, the ratio of the biomarker concentration to the total protein concentration of the megakaryocyte derivative is preferably different from the ratio of the same biomarker concentration to the total protein concentration of PRGF. For example, in some embodiments, the ratio of the EGF concentration to the total protein concentration of the megakaryocyte derivative (e.g., MLC, PLC, their derivatives, or their lysates) is preferably higher than the ratio of the EGF concentration to the total protein concentration of PRGF. In some embodiments, the ratio of the PDGF-BB concentration to the total protein concentration of the megakaryocyte derivative (e.g., MLC, PLC, their derivatives, or their lysates) is preferably higher than the ratio of the PDGF-BB concentration to the total protein concentration of PRGF. 【0153】 Compositions comprising megakaryocyte derivatives as described herein are provided herein. In some embodiments, the composition further comprises one or more further therapeutic agents. In some embodiments, one or more further therapeutic agents include wound healing agents, tissue regeneration agents, anti-apoptotic agents, anti-inflammatory agents, neurotropic agents, anti-hormonal agents or immunomodulators or combinations thereof. In some embodiments, the composition does not contain red blood cells or hemoglobin contents or white blood cells. In some embodiments, the tissue regeneration agent is (a) a growth factor selected from one or more of the following: transforming growth factor (TGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet-derived endothelial growth factor (PDEGF), platelet-derived angiogenic factor (PDAF), platelet factor 4 (PF-4), hepatocyte growth factor (HGF), or a combination thereof; and (b) one or more of the following cytokines selected from one or more of the following: IL-1B, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17A, IL-23, TNF-alpha, or a combination thereof. In some embodiments, administration of a therapeutic dose of the composition results in a statistically significant reduction in injury recovery as measured after administration of the composition, compared to administration of the vehicle alone. In some embodiments, administration of a therapeutic dose of the composition results in a statistically significant improvement in one or more of the following, measured after administration of the composition: pain, stiffness, and function, compared to administration of the vehicle alone. 【0154】 【0155】 It may be desirable to administer other compounds to patients, such as corticosteroids, tetrasubstituted pyrimidopyrimidines, glucocorticoids, beta-catenin proteins or polypeptides or their agonists, NSAIDs (e.g., naproxen sodium, diclofenac sodium, diclofenac potassium, aspirin, sulindac, diflunisal, piroxicam, indomethacin, ibuprofen, nabumetone, choline magnesium trisalicylate, sodium salicylate, salicylsalicylic acid, fenoprofen, flurbiprofen, ketoprofen, sodium meclofenamate, meloxicam, oxaprozin, sulindac, and tolmetin), COX-2 inhibitors (e.g., rofecoxib, celecoxib, valdecoxib, and lumiracoxib), glucocorticoid receptor modulators, or DMARDs. The combination therapies described herein are particularly useful for treating immunoinflammatory disorders in combination with other agents, biological agents, or small molecules that modulate the immune response and have a positive effect on the disease. Such agents include those that deplete key inflammatory cells, affect cell adhesion, or affect cytokines involved in the immune response. This last category includes both agents that mimic or increase the action of anti-inflammatory cytokines such as IL-10, and agents that inhibit the activity of pro-inflammatory cytokines such as IL-1B, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17A, IL-23, MIG, MCP-1, IP-10, or TNF-alpha. Examples of agents that inhibit TNF-alpha include etanercept, adelimumab, infliximab, and CDP-870. In this example (an example of a drug that blocks the action of TNF-alpha), the combination therapy reduces cytokine production, and etanercept or infliximab acts on the remaining inflammatory cytokines, providing enhanced treatment.Examples of small molecule immunomodulators include p38 MAP kinase inhibitors such as VX 702, SCIO 469, Drampimod, RO 30201195, and SCIO 323; TACE inhibitors such as DPC 333; ICE inhibitors such as Pranarcasan; and IMPDH inhibitors such as mycophenolate and melimepodiv. 【0156】 Other therapeutic agents that can be administered before, simultaneously with, or after a gap in administration with megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom) include thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocon, metsuredopa, and uredopa; altoretamine, triethylenemelamine, and triethylenephosphoramide. , including triethylenethiophosphoramide and trimethylolmelamine, ethyleneimine and methylamelamine; acetogenins (especially bratacin and bratacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapacon; lapachol; colchicine; betulinic acid; camptothecin (synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9 -Containing aminocamptothecin); bryostatin; calistatin; CC-1065 (including its adzeresin, karzeresin, and bizeresin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (including synthetic analogs, KW-2189 and CB1-TM1); eloiterobin; pancratistatin; sarcodictyin; spongstatin; chlorambucil, chlornafadin, chlorofo Nitrogen mustards such as sphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenestrine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; engine antibiotics (e.g., calichemycin, especially calichemycin gamma 1 and calichemycin omega 2); and the oral alpha-4 integrin inhibitor CDP323;Dynemicin A (including dynemicin; esperamicin; and neocardinostatin chromophore and related pigment protein enediin antibiotic chromophore), acrasinomycin, actinomycin, ausramycin, azaserin, bleomycin, kactinomycin, carabicin, kaminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®), liposomal doxorubicin TLC) Antibiotics such as D-99 (MYOCET®), pegylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zorubicin; methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epotilon, and others. Antimetabolites such as 5-fluorouracil (5-FU); combretastatin; folic acid analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid; acegraton;Aldophosphamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatraxate; defofamine; demecoltin; diaziquan; eformithine; eriptinium acetate; epotilon; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; mayansinoids such as mayansine and ansamitosine; mitogluazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK (trademark) polysaccharide complex (JHS Natural) Products, Eugene, Oreg); Lazoxane; Rhizoxin; Sizofuran; Spirogermanium; Tenuazonic Acid; Triadiquan; 2,2',2'-Trichlorotriethylamine; Trichothecene (especially T-2 toxin, verracurin A, loridine A and anguidin); Urethane; Vindesine (ELDISINE®, FILDESIN®); Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Thiotepa; Taxoids, e.g., Paclitaxel (TAXOL®, Bristol-Myers Squibb) (Oncology, Princeton, NJ), albumin-modified nanoparticle formulations of paclitaxel (ABRAXANE®), and docetaxel (TAXOTERE®, Rhome-Poulene Rorer, Antony, France); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vinca, including, for example, vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®), which prevent tubulin polymerization from forming microtubules;Etoposide (VP-16); Ifosfamide; Mitoxantrone; Leucovorin; Novantrone; Edatrexate; Daunomycin; Aminopterin; Ibandronate; Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids such as retinoic acid containing bexarotene (TARGRETIN®); Clodronate (e.g., BONEFOS® or OSTAC®), Etidronate (DIDROCAL®), NE-58095, Zoledronic acid / Zoledrone (ZOMETA®), Alendronate (FOSAMAX®), Pamidronate (AREDIA®), Childronate (SKELID®), or bisphosphonates such as risedronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, in particular those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R) (e.g., erlotinib (Tarceva®)); and VEGF-A that reduces cell proliferation; THERAT Vaccines such as OPE(trademark) vaccines and gene therapy vaccines, e.g., ALLOVECTIN(trademark) vaccine, LEUVECTIN(trademark) vaccine, and VAXID(trademark) vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN(trademark)); rmRH (e.g., ABARELIX(trademark)); BAY439006 (sorafenib, Bayer); SU-11248 (sunitinib, SUTENT(trademark), Pfizer); perifosine, COX-2 inhibitors (e.g., Bcl-2 inhibitors such as celecoxib or etoricoxib, proteosome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); olafenib (orafenib), ABT510; oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors; tyrosine kinase inhibitors; serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®);Examples include farnesyltransferase inhibitors such as ronafarnib (SCH 6636, SARASAR); and any pharmaceutically acceptable salts, acids, or derivatives of the above; as well as CHOP, an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for treatment regimens with oxaliplatin (ELOXATIN®) in combination with 5-FU and leucovorin; and any pharmaceutically acceptable salts, acids, or derivatives of the above; and two or more combinations of the above. 【0157】 Therapeutic agents as defined herein include “anti-hormone agents” or “endocrine agents” that act to modulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth. Therapeutic agents include, but are not limited to, anti-estrogen and selective estrogen receptor modulators (SERMs) such as tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; hormones themselves; for example, 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozol, RIVISOR® borozole, FEMARA® letrozole, and ARIMIDESEX® anastrozole; and flutamide, nilutamide, bicalutamide. Antiandrogens such as leuprolide and goserelin; as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, in particular those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, and H-Ras; ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME® ribozyme) and HER2 expression inhibitors; vaccines such as gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rlL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; vinorelbine and esperamicin; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above; as well as any combination of two or more of the above. Immunomodulatory drugs 【0158】 In some embodiments, the Disclosure also includes immunomodulatory agents for use with megakaryocyte derivatives of the Disclosure (e.g., MLC, PLC, their derivatives or lysates) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC, their derivatives or lysates, or PRP, PRGF, or AS derived therefrom). The term “immunomodulatory agent” refers to a type of drug that modifies an immune system response or function of the immune system, for example, by stimulating antibody formation and / or inhibiting peripheral blood cell activity, and includes, but is not limited to, thalidomide (aN-phthalimide-glutalimid) and its analogues, REVLIMID® (lenalidomide), ACTI-MID® (pomaridomide), OTEZLA® (apremilast), and pharmaceutically acceptable salts or acids thereof. 【0159】 The therapies described herein may be administered alone or in combination with other therapies, and may be provided at home, in a clinic, medical practice, outpatient setting, or in a hospital. Treatment may be initiated in a hospital setting, or it may be initiated on an outpatient basis, as necessary, to allow a physician to closely monitor the effects of the therapy and make necessary adjustments. The duration of the therapy will depend on the type of disease or disorder being treated, the patient's age and condition, the stage and type of the patient's disease, and how the patient responds to the treatment. If necessary, individuals at higher risk of developing inflammatory diseases (e.g., those undergoing age-related hormonal changes) may receive treatment to inhibit or delay the onset of symptoms. 【0160】 In combination therapy, the dosage and frequency of administration of each component of the combination can be controlled independently. For example, one compound may be administered once, twice, or three times per day, week, or month, while a second compound may be administered once per day, week, or month. Combination therapy may be administered in on-off cycles, including rest periods, to allow the patient's body to recover from any previously unforeseen side effects. The compounds may also be formulated together so that a single dose delivers both compounds. Except when administered simultaneously, one component in combination therapy may be administered within hours, days, or weeks of the other component, depending on the patient's desired administration needs. 【0161】 In some embodiments, 10 6 Less than 10 PLCs or about 10 6 ~10 pieces 7 10 PLCs or about 10 7 ~10 pieces 8 10 PLCs or about 10 8 ~10 pieces 9 Individual PLCs or about 10 9 ~10 pieces 10 10 PLCs or 10 10 More than (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10¹ units are administered to the target. For example, approximately (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10¹ units. 6 (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 7 10 PLCs, approximately (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 7 PLCs (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 8 10 PLCs, or approximately (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 8 PLCs (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 9 10 PLCs or approximately (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 9 PLCs (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 10 Individual PLCs may be administered to the target. 【0162】 In some embodiments, 10 6 Less than 10 MLCs or about 10 6 ~10 pieces 7 Individual MLCs or about 10 7 ~10 pieces 8 Individual MLCs or about 10 8 ~10 pieces 9 Individual MLCs or about 10 9 ~10 pieces 10 10 MLCs or 10 10 More than 10 MLCs are administered to the target. For example, approximately (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 6 (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 7 Each MLC, approximately (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 7 Individual MLCs (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 8 10 MLCs, or approximately (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 8 Individual MLCs (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 9 10 MLC or approximately (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 9 Individual MLCs (1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 10 Individual MLCs may be administered to the target. 【0163】 This disclosure provides a kit comprising megakaryocyte derivatives for use according to this disclosure (e.g., MLC, PLC or derivatives thereof or lysates thereof, or PRP, PRGF, or AS derived therefrom) (i.e., lysates derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC or PLC or derivatives thereof, or PRP, PRGF, or AS derived therefrom), and optionally a carrier, buffer, emulsifier, or excipient, the kit further comprising instructions for use for administration to a subject. The kit may optionally further contain one or more drugs in one or more containers. The kit may further comprise a label or accompanying information sheet. The accompanying information sheet provides instructions for use, dosage instructions, administration, contraindications, and / or warnings regarding the use of such contents of the kit. Examples of containers include vials, bottles, syringes, blister packs, etc. The active drug (e.g., MLC or PLC or derivatives thereof or lysates thereof, or combinations thereof, or any further drugs contained in the kit) can usually be stored as a solid composition, a lyophilized formulation, or an aqueous solution. 【0164】 The following examples are presented to provide a complete disclosure and description of the assays, screenings, and methods for preparing and using the therapeutic agents of this disclosure, and are not intended to limit the scope of what the inventors consider to be the disclosure. Manufacturing of megakaryocyte derivatives 【0165】 Methods for producing megakaryocyte derivatives are described herein. In some embodiments, the method comprises (a) culturing a population of progenitor cells ex vivo for a period of time during which the progenitor cells differentiate into mature megakaryocytes; (b) isolating a population of MLCs, PLCs, or their derivatives from the megakaryocytes; and (c) enriching the MLCs, PLCs, or their derivatives. In some embodiments, the method further comprises lysing the MLCs, PLCs, or their derivatives. In some embodiments, the method further comprises enriching the lysed MLCs, PLCs, or their derivatives. In some embodiments, the method further comprises mixing with donor-derived PRP. In some embodiments, the progenitor cells include human induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), mesenchymal stem / stromal cells (MSCs), hematopoietic stem cells, immortalized megakaryocyte progenitor cells, and CD34 cells. + Umbilical cord blood stem cells (UCB cells), CD34 + Mobilized peripheral blood cells (MPB cells) or CD34 + One or more cells are selected from among bone marrow cells. In some embodiments, the culture of the progenitor cell population ex vivo is carried out in a bioreactor. In some embodiments, MLC, PLC, or derivatives thereof are isolated from megakaryocytes by a pressure gradient. In some embodiments, the pressure gradient is generated in a bioreactor. In some embodiments, megakaryocyte derivatives, PLC, MLC, MLC lysates, and / or derivatives thereof are prepared using any of the methods described in U.S. Patent No. 11,400,118 and No. 10,426,799, and / or U.S. Patent Application Publication No. 16 / 957,918, which are incorporated herein by reference in whole. 【0166】 Alternatively, in some embodiments, the method comprises (a) culturing a population of somatic cells, progenitor cells, stem cells, or combinations thereof ex vivo for a period of time to differentiate into mature megakaryocytes; (b) isolating a population of MLCs, PLCs, or derivatives thereof from megakaryocytes by a pressure gradient in a bioreactor; and (c) enriching the MLCs, PLCs, or derivatives thereof. In some embodiments, the method further comprises lysing the MLCs, PLCs, or derivatives thereof. In some embodiments, the method further comprises enriching the lysed MLCs, PLCs, or derivatives thereof. In some embodiments, the method further comprises mixing with donor-derived PRP. In some embodiments, the progenitor cells include human induced pluripotent stem cells (iPSCs), hematopoietic stem cells, embryonic stem cells (ESCs), immortalized megakaryotic progenitor cells, and CD34 cells. + Umbilical cord blood stem cells (UCB cells), CD34 + Mobilized peripheral blood cells (MPB cells) or CD34 + One or more cells are selected from bone marrow cells. In some embodiments, the culture of the progenitor cell population ex vivo is carried out in a bioreactor. In some embodiments, MLCs, PLCs, or their derivatives are isolated from megakaryocytes by a pressure gradient. In some embodiments, the pressure gradient is generated in a bioreactor. Exemplary Embodiment 【0167】 The methods and compositions of this disclosure preferably utilize stem cell-derived megakaryocyte derivatives (e.g., megakaryocyte-like cell (MLC) derivatives or derivatives thereof, or lysates thereof, or platelet-rich plasma derived therefrom, such as novel, anucleated platelets or platelet-like cells or platelet variants (collectively referred to as "PLCs") (or "PLCs" in the singular form)) to meet unmet needs for treating, repairing, or restoring dry eye-related diseases, disorders, or injuries for which appropriate or consistent treatment is unavailable by conventional means. 【0168】 Preferably, megakaryocyte derivatives (e.g., MLC, PLC, their lysates, or their derivatives) have consistent quality and composition and can be produced at a relatively low cost compared to those obtained from human donor blood. More importantly, megakaryocyte derivatives eliminate the need to puncture patients to collect blood. Furthermore, megakaryocyte derivatives (e.g., MLC or PLC) are easy to prepare as they emerge from bioreactors or fluid devices and can be administered in a less aggressive manner than other treatment options (e.g., without surgery or complex medical procedures). Megakaryocyte derivatives (e.g., MLC, PLC, their lysates, or their derivatives) produced by the methods of this disclosure can be easily scaled up, have a potentially continuous supply (without being hindered by batch-to-batch variability), are relatively free of impurities, can be used to supplement donor-derived PRP, PRGF, or AS, and can be administered topically at or near the site of injury to treat, repair, or alleviate dry eye disease. 【0169】 Accordingly, in some embodiments, compositions are provided that include megakaryocyte derivatives (e.g., MLC, PLC, or their derivatives or lysates thereof, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC, or their derivatives, or PRP, PRGF, or AS derived therefrom) which can be administered directly to or near the eye. In some embodiments, compositions containing megakaryocyte derivatives (e.g., MLC, PLC, or their lysates or their derivatives, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC, or their lysates or their derivatives, or PRP, PRGF, or AS derived therefrom) are administered topically or locally in, around, and below the eye. In some embodiments, megakaryocyte derivatives (e.g., MLC, PLC, their lysates, or derivatives thereof) or precursor cells for producing megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or PRP, PRGF, or AS derived therefrom) are in the form of lysates, i.e., megakaryocyte derivatives (e.g., MLC, PLC, or their derivatives) or precursor cells for producing megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or PRP, PRGF, or AS derived therefrom) are concentrated, and the lysates are prepared from the concentrate. In some embodiments, the PLC or lysates are concentrated and diluted before use in a carrier, diluent, or buffer, or in donor-derived PRP, PRGF, or AS as disclosed herein. 【0170】 In some embodiments, megakaryocyte derivatives (e.g., MLC, PLC, their derivatives, or their lysates) are concentrated together with growth factors, or with agents that stimulate the release of growth factors from the megakaryocyte derivatives (e.g., MLC, PLC, their lysates, or their derivatives), or are mixed with platelet-rich plasma derived from the subject in a combination optimal for the subject. 【0171】 Megakaryocyte derivatives (e.g., MLCs, PLCs, their lysates, or their derivatives) are produced by one or more tools and techniques, such as bioreactors or fluid devices. Examples of bioreactors or fluid devices include, but are not limited to, shear stress, mechanical strain, and pulsed electromagnetic field bioreactors, large-scale agitated tank bioreactors, automated bioreactors, rotating wall bioreactors (RWBs), and oscillating motions as seen in wave bioreactors, and organ-on-chip bioreactors. Other bioreactor configurations that enable continuous perfusion operations, such as packed-bed bioreactors (PBBs), fluidized-bed bioreactors (FBBs), or membrane bioreactors including PBBs or FBBs using microcarriers, CultiBag bioreactors, and hollow fiber bioreactors (HFBs), are also intended to produce the PLCs / EVs of this disclosure or their derivatives. Bioreactor operations may require coupling with internal or external cell retention devices in a recycling line, using in vivo bioreactors, which are pockets within the body where biomaterials (e.g., PLC or its derivatives or progenitor cells derived from them) are implanted at the site where they are needed and incubated for an extended period. Within these pockets (e.g., eyes, glands), grafts utilize the regenerative capacity of the body to recover from disease or injury.A non-limiting example of a bioreactor is, for example, Simultaneous Welding of Three Components to Form a Bioreactor or Filter. Tools and technologies (e.g., bioreactors or fluid devices) are described in the joint application titled Structure (U.S. Patent Application Publication No. 62 / 981,373) or elsewhere disclosed in U.S. Patents No. 9,795,965, No. 10,343,163, No. 9,763,984, No. 9,993,503, and No. 10,426,799; U.S. Patent Application Publication No. 20180334652; PCT Application No. PCT / US2018 / 021354; PCT / US2019 / 012437, PCT / US19 / 040021, and U.S. Patent Application Publication No. 16 / 730,603, each of which is incorporated herein by reference in whole. Known or unknown bioreactors or microfluidic devices capable of routinely generating MLCs, PLCs, or derivatives are also intended for use in this disclosure. 【0172】 In some embodiments, the source of megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or lysates derived therefrom, or PRP, PRGF, or AS derived therefrom), or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC, their lysates or their derivatives, or PRP, PRGF, or AS derived therefrom), is the individual itself, i.e., they are, for example, the CD34 of an individual requiring megakaryocyte derivative-based treatment (e.g., MLC-based treatment, PLC-based treatment). +Progenitor cells are used for production, where the progenitor cells are cultured in a bioreactor to produce megakaryocyte derivatives (e.g., MLC, PLC, their lysates, or their derivatives), which can be used by themselves, i.e., megakaryocyte derivatives (e.g., MLC, PLC, or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom (i.e., PRP, PRGF, or AS concentrated with MLC, PLC, their lysates, or their derivatives)). In some embodiments, the source of the megakaryocyte derivatives (e.g., MLC, PLC, or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom) is iPSC or megakaryocytes derived from iPSC. 【0173】 Some other advantages offered by methods and compositions comprising megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) are that they are essentially allogeneic, minimizing the risk of immune response, are non-cancerous, i.e., do not exhibit uncontrolled proliferation or tumorigenesis in vivo, and are rich in growth factors such as fibronectin, vitronectin, and sphingosine 1-phosphate, which promote the healing process. Megakaryocyte derivatives (e.g., MLC or PLC or their derivatives, or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) can also reduce or eliminate adverse side effects induced by, for example, anti-inflammatory agents, opioids, or other drugs. 【0174】 In some embodiments, the Disclosure provides a method for treating a subject suffering from an injury, the method comprising administering a therapeutic dose to the subject a composition comprising a megakaryocyte derivative of the Disclosure (e.g., MLC, PLC or derivatives thereof or lysates thereof, or PRP, PRGF, or AS derived therefrom), thereby resulting in recovery, treatment, or repair of the injury (e.g., dry eye disease). In some embodiments, the method comprises administering a second or third therapeutic agent. 【0175】 In some embodiments, the disclosure provides a pharmaceutical composition comprising a megakaryocyte derivative (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing a megakaryocyte derivative (e.g., MLC, PLC or its derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) and one or more pharmaceutically acceptable expanders, carriers, or excipients. In some embodiments, the pharmaceutical composition further comprises a second or third therapeutic agent. 【0176】 In some embodiments, this disclosure provides non-native extracellular vesicles (EVs) prepared in vitro as a mixture with megakaryocyte derivatives (e.g., MLCs, PLCs, their lysates, or their derivatives). EVs comprise microvesicles (MVs) 200–1000 nm in diameter and exosomes, each harboring a wide variety of biologically active molecules, such as proteins, lipids, and RNA, either on their surface or within their lumen. Each component in the mixture, i.e., megakaryocyte derivatives (e.g., MLCs, PLCs, their lysates, or their derivatives), microvesicles, and exosomes, can be substantially isolated from the mixture into individual components, for example, based on their size. Extracellular vesicles (EVs) have been shown to play a role in stimulating eye healing, for example, by conferring eye healing, anti-apoptotic, or anti-inflammatory effects through the transport of RNA and protein cargo. Extracellular vesicles also function as transport and delivery systems for bioactive molecules, playing roles in hemostasis and thrombosis, inflammation, malignant tumor infection transport, angiogenesis, and immunity. Therefore, in some embodiments, EVs may complement megakaryocyte derivatives (e.g., MLC, PLC, their lysates, or their derivatives), and the use of combinations thereof represents an even richer resource for megakaryocyte derivative-based therapeutic applications (e.g., MLC-based therapeutic applications, PLC-based therapeutic applications). 【0177】 In some embodiments, the extracellular matrix (EV) of this disclosure comprises exosomes ranging in diameter from approximately 65 nm to approximately 10 μm, harboring a wide variety of molecules, such as proteins, lipids, and RNA, either on their surface or within their lumen. Exosomes have been shown to play a role in stimulating tissue regeneration in many in vitro and in vivo models, and they can confer pro-angiogenic, proliferative, anti-apoptotic, and anti-inflammatory effects through the transport of RNA and protein cargo. Therefore, in some embodiments, exosomes become an even richer resource for megakaryocyte derivative-based therapeutic applications (e.g., MLC-based therapeutic applications, PLC-based therapeutic applications). In some embodiments, megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or their lysates, or PRP, PRGF, or AS derived therefrom) or precursor cells for producing megakaryocyte derivatives (e.g., MLC, PLC, their lysates, or their derivatives, or PRP, PRGF, or AS derived therefrom) are produced as a mixture with the megakaryocyte derivatives (e.g., MLC, PLC, their lysates, or their derivatives), but are administered in combination with extracellular vesicles (EVs) that can be substantially isolated from the megakaryocyte derivatives (e.g., MLC, PLC) based on their smaller size. Therefore, in one embodiment, microvesicles or exosomes can be used alone or in combination with megakaryocyte derivatives (e.g., MLC, PLC, their lysates, or derivatives thereof) to stimulate the healing of dry eye diseases, for example, through actions such as the transport of RNA or transporters (e.g., eye healers, anti-apoptotics, anti-inflammatorys, etc.) or protein cargo, thereby becoming an even richer resource for megakaryocyte derivative-based therapeutic applications (e.g., MLC-based therapeutic applications, PLC-based therapeutic applications) to confer potentially desirable pro-angiogenic, proliferative, anti-apoptotic, or anti-inflammatory effects in patients receiving PLC-based treatments as disclosed herein. 【0178】 In some embodiments, megakaryocyte progenitor cells, megakaryocytes, platelet precursors, and preplatelets derived from induced pluripotent stem cells (iPSCs) that produce megakaryocyte-like cells (MLCs), platelet-like cells (PLCs), and EVs (i.e., microvesicles or exosomes or combinations thereof) may be genetically engineered to express nucleic acids encoding a protein of interest (e.g., eye healers, anti-apoptotics, anti-inflammatory agents, etc.) before passing through a bioreactor or fluid device. In some embodiments, MLCs, PLCs, and / or EVs may be genetically engineered once such cells are subjected to passage through a bioreactor or fluid device. Thus, in some embodiments, genetic modification may be performed at the stem cell level, in megakaryocytes, or in some embodiments, in PLCs and / or EVs, or at any other level during the generation of MLCs, PLCs, and / or EVs associated with the production of MLCs, PLCs, and / or EVs. Genetic engineering of megakaryocytes or megakaryocyte progenitor cells differentiated from genetically engineered human pluripotent stem cells (hPSCs) or cell lines is also intended by this disclosure, where the genetic engineering causes the megakaryocytes or megakaryocyte progenitor cells to express a protein or polypeptide of interest. In some embodiments, MLCs, PLCs and / or EVs, or derivatives thereof, differentiated from genetically engineered progenitor cells (e.g., megakaryocytes or megakaryocyte progenitor cells), deliver the protein of interest (e.g., an eye healer, an anti-apoptotic agent, an anti-inflammatory agent, etc.) systemically, or at a first site of disease, generally the site of the disease where the MLCs, PLCs and / or EVs (or their genetically engineered versions) are administered, or at a second site of disease different from the site where the MLCs, PLCs and / or EVs, or their derivatives, are administered. Examples of megakaryocytes derived from such genetically engineered induced pluripotent stem cells or PSCs that produce PLC and / or EV (i.e., genetically engineered PLC / EV or derivatives thereof) are disclosed in concurrently pending U.S. Patent Applications Publications 17 / 213,552 and 17 / 213,796, which are incorporated herein by whole reference, respectively.Therefore, in some embodiments, genetically engineered PLC (ePLC) may be produced by genetically engineered PLC-producing progenitor cells so that ePLC expresses an exogenous gene of interest, such as an eye healer, anti-apoptotic agent, anti-inflammatory agent, anti-hormone agent, or immunomodulator, and its enrichment broadly complements PLC-based treatments. 【0179】 It may be desirable to administer other compounds to patients, including but not limited to corticosteroids, tetrasubstituted pyrimidopyrimidines, NSAIDs (e.g., naproxen sodium, diclofenac sodium, diclofenac potassium, aspirin, sulindac, diflunisal, piroxicam, indomethacin, ibuprofen, nabumetone, magnesium choline trisalicylate, sodium salicylate, salicylsalicylic acid, fenoprofen, flurbiprofen, ketoprofen, sodium meclofenamate, meloxicam, oxaprozin, sulindac, and tolmetin), COX-2 inhibitors (e.g., rofecoxib, celecoxib, valdecoxib, and lumiracoxib), glucocorticoid receptor modulators, or DMARDs. The combination therapies described herein are particularly useful for treating immunoinflammatory disorders in combination with other agents, biological agents, or small molecules that modulate the immune response and have a positive effect on disease treatment. Such drugs include those that deplete key inflammatory cells, affect cell adhesion, or influence cytokines involved in the immune response. This last category includes both drugs that mimic or increase the action of anti-inflammatory cytokines such as IL-10, and drugs that inhibit the activity of pro-inflammatory cytokines such as IL-1B, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17A, IL-23, MIG, MCP-1, IP-10, or TNF-alpha. Examples of TNF-alpha inhibitors include etanercept, adelimumab, infliximab, and CDP-870. In this example (an example of a drug that blocks the action of TNF-alpha), combination therapy reduces cytokine production, and etanercept or infliximab acts on the remaining inflammatory cytokines, providing enhanced treatment. Examples of small molecule immunomodulators include p38 MAP kinase inhibitors such as VX 702, SCIO 469, Drampimod, RO 30201195, and SCIO 323; TACE inhibitors such as DPC 333; ICE inhibitors such as Pranarcasan; and IMPDH inhibitors such as mycophenolate and melimepodiv.Preferably, when one or more of these compounds are administered together with megakaryocyte derivatives (e.g., MLC, PLC, their lysates, or derivatives thereof), the concentration of the co-administered compounds, if included in co-therapy, is reduced or decreased, thereby reducing any known and unknown adverse side effects that would be induced by the co-administered compounds if they were administered without megakaryocyte derivatives (e.g., MLC, PLC, their lysates, or derivatives thereof). 【0180】 In some embodiments, the Disclosure provides a kit comprising megakaryocyte derivatives of the Disclosure (e.g., MLC, PLC or derivatives thereof, or lysates thereof, or PRP, PRGF, or AS derived therefrom) or precursor cells (e.g., MLC, PLC, lysates thereof, or derivatives thereof, or PRP, PRGF, or AS derived therefrom) for producing megakaryocyte derivatives. 【0181】 In some embodiments, the techniques described herein relate to methods for treating, repairing, or restoring a condition in a subject requiring such treatment, comprising administering to the subject an effective amount of a treatment composition containing a megakaryocyte derivative (e.g., megakaryocyte-like cells (MLCs), platelet-like cells (PLCs) or derivatives or lysates thereof, or PRP, PRGF, or AS derived therefrom), wherein the condition is dry eye disease and the route of administration is ophthalmoscopy or intraglandular. 【0182】 In some embodiments, the techniques described herein relate to a method for producing megakaryocyte derivatives (e.g., MLC or PLC) containing PRP, the method comprising: culturing a population of progenitor cells ex vivo in a bioreactor for a period of time during which the progenitor cells are differentiated into mature megakaryocytes; isolating a population of MLC, PLC, or their derivatives or lysates, or PRP, PRGF, or AS derived therefrom, separated from megakaryocytes by a pressure gradient in the bioreactor; concentrating the MLC, PLC, or their derivatives or lysates, or PRP, PRGF, or AS derived therefrom; optionally lysing the MLC, PLC, or their derivatives or lysates, or PRP, PRGF, or AS derived therefrom; and optionally mixing them with donor-derived PRP. 【0183】 In some embodiments, the techniques described herein relate to methods for treating dry eye disease in patients using megakaryocyte derivatives (e.g., MLC derivatives, e.g., MLC lysates, in vitro anucleated populations of platelet-like cells (PLC) or their derivatives or lysates, or PRP, PRGF, or AS derived therefrom) that have the following characteristics: i) derived from the reprogramming of somatic cells, progenitor cells, or stem cells, the product passes through a bioreactor; ii) are not cancerous cells; and iii) do not exhibit uncontrolled proliferation or tumorigenesis in vivo, wherein the megakaryocyte derivatives (e.g., MLC, PLC or their derivatives or lysates, or PRP, PRGF, or AS derived therefrom) are administered in therapeutic doses to patients requiring such treatment, and the route of administration is by eye drops, intraglandular, subconjunctival, topical, sub-Tenon, intravitreous, or anterior chamber. 【0184】 In some embodiments, the techniques described herein relate to a method for treating dry eye disease in a subject requiring such treatment, comprising administering to the subject an effective amount of a composition comprising a megakaryocyte derivative (e.g., MLC, PLC or derivatives or lysates thereof, or PRP, PRGF, or AS derived therefrom), wherein the composition is administered in a therapeutic dose, a statistically significant reduction in injury recovery is measured after administration of the composition compared to administration of the vehicle alone, and / or a statistically significant improvement in one or more of pain, stiffness, and function is measured after administration of the composition compared to administration of the vehicle alone, and the route of administration is by eye drops, intraglandular, subconjunctival, topical, sub-Tenon, intravitreous, or anterior chamber. 【0185】 In some embodiments, the techniques described herein relate to methods for treating, repairing, or restoring dry eye disease in subjects requiring such treatment, comprising administering to the subject multiple doses of an effective amount of a treatment composition containing a megakaryocyte derivative (e.g., megakaryocyte-like cells (MLCs), platelet-like cells (PLCs) or derivatives or lysates thereof, or PRP, PRGF, or AS derived therefrom), wherein the route of administration is ophthalmos or intraglandular. 【0186】 In some embodiments, the techniques described herein relate to methods for treating, repairing, or restoring dry eye disease in subjects requiring such treatment, comprising administering to the subject multiple doses of an effective amount of a treatment composition containing a megakaryocyte derivative (e.g., megakaryocyte-like cells (MLCs), platelet-like cells (PLCs) or derivatives or lysates thereof, or PRP, PRGF, or AS derived therefrom), wherein the disease is not cancer, the treatment is not for cancer, and the route of administration is by eye drops, intraglandular, subconjunctival, topical, sub-Tenon's, intravitreous, or anterior chamber. 【0187】 In some embodiments, the techniques described herein relate to compositions for treating dry eye disease in subjects, the compositions comprising megakaryocyte derivatives (e.g., MLC, PLC or derivatives or lysates thereof, or PRP, PRGF, or AS derived therefrom), wherein the route of administration is by ophthalmos, intraglandular, subconjunctival, topical, sub-Tenon, intravitreous, or anterior chamber. 【0188】 Methods for treating, repairing or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, the methods comprising administering to the subject an effective amount of a composition comprising a megakaryocyte derivative, wherein the route of administration is by eye drops or intraglandular instillation, and the megakaryocyte derivative comprises MLC lysates, PLC, exosomes, megakaryocytes, or a combination thereof. Furthermore, methods for treating, repairing or restoring dry eye disease in subjects requiring such treatment are provided herein, wherein the composition further comprises wound healing agents, tissue regeneration agents, anti-apoptotic agents, anti-inflammatory agents, neurotropic agents, anti-hormonal agents or immunomodulators, or a combination thereof. Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment for dry eye disease are provided herein, wherein the tissue regenerating agent is (a) a growth factor selected from one or more of the following: transforming growth factor (TGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet-derived endothelial growth factor (PDEGF), platelet-derived angiogenic factor (PDAF), platelet factor 4 (PF-4), hepatocyte growth factor (HGF), or a combination thereof; and (b) one or more of the following cytokines selected from one or more of the following: IL-1B, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17A, IL-23, TNF-alpha, or a combination thereof.Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment for dry eye disease are provided herein, wherein megakaryocyte derivatives include fibroblast growth factor-2 (FGF-2); hepatocyte growth factor (HGF); insulin-like growth factor 1 (IGF-1); activated regulated normal T cell expression secretion factor (RANTES); neuronal growth factor (NGF); vascular endothelial growth factor (VEGF-A); vascular endothelial growth factor (VEGF-C); epidermal growth factor (EGF); transforming growth factor-β1 (TGF-β1); transforming growth factor-β2 (TGF-β1); The biomarker comprises one or more biomarkers selected from platelet-derived growth factor-AA (PDGF-AA); platelet-derived growth factor-BB (PDGF-BB); platelet-derived growth factor-AA / BB (PDGF-AA / BB); interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-12p40 (IL-12p40); interleukin-12p70 (IL-12p70); tissue metalloproteinase inhibitor 1 (TIMP-1); tissue metalloproteinase inhibitor 2 (TIMP-2); and tissue metalloproteinase inhibitor 3 (TIMP-3). Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the megakaryocyte derivative comprises an EGF concentration per unit total protein that is higher than the mean EGF concentration per unit total protein measured in human platelets, platelet-rich plasma (PRP), or multi-growth factor plasma (PRGF), or in a range of EGF concentrations higher than the corresponding EGF concentration in the total protein concentration of PRGF. Furthermore, methods for treating, repairing or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the composition further comprises a lysate derived from the subject, serum, plasma, multi-growth factor plasma (PRGF), platelet-rich plasma (PRP), or any other blood derivative. Furthermore, methods for treating, repairing or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the route of administration is subconjunctival or topical.Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the route of administration is sub-Tenon. Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the route of administration is intravitreous or intrachorium. Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the dry eye disease is caused by Sjögren's syndrome or non-Sjögren's syndrome. Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the megakaryocyte derivative does not contain erythrocytes or hemoglobin contents or leukocytes. Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the composition further comprises extracellular vesicles (EVs). Furthermore, methods for treating, repairing or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the composition is formulated for application to a site of injury or tissue damage for therapeutic use. Furthermore, methods for treating, repairing or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the composition is formulated in a buffer, diluent, or excipient, or a combination thereof. Furthermore, methods for treating, repairing or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the composition comprises a) 0.01 to 100% by weight of a megakaryocyte derivative, b) 0 to 90% by weight of an expander, and / or c) 0 to 90% by weight of at least one excipient or carrier, and optionally d) platelet-rich plasma (PRP), PRGF, plasma, serum, or other blood derivatives derived from the subject. Furthermore, methods for treating, repairing or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the composition is lyophilized.Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the composition is implanted on an implantable device. Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the composition is cryopreserved. Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the composition is administered topically to one or more sites or vicinity of the injury or disease. Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the composition further comprises another therapeutic agent. Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the megakaryocyte derivative is derived from the reprogramming of somatic cells, progenitor cells, or stem cells. Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the megakaryocyte derivative is not a cancerous cell. Furthermore, methods for treating, repairing or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the megakaryocyte derivative does not exhibit uncontrolled proliferation or tumorigenesis in vivo. Furthermore, methods for treating, repairing or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the method reduces injury recovery time. Furthermore, methods for treating, repairing or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein the method improves one or more of the pain, stiffness and function of the eye, or one or more signs and / or symptoms of dry eye disease, or one or more of the pain, discomfort, visual function, ocular surface disease index, corneal fluorescein score, tear production, tear quality, tear breakdown time, immune cell infiltration, goblet cell count, mucin levels, etc.Furthermore, methods for treating, repairing, or restoring dry eye disease in subjects requiring treatment of dry eye disease are provided herein, wherein megakaryocyte derivatives are fabricated in a fluid device or bioreactor. 【0189】 A method for treating, repairing, or restoring a condition in a subject requiring treatment is provided herein, comprising administering to the subject a plurality of doses of an effective amount of a composition containing a megakaryocyte derivative, wherein the condition is selected from dry eye disease, ocular surface disease, osteoarthritis, and wound healing. Furthermore, a method for treating, repairing, or restoring a condition in a subject requiring treatment is provided herein, wherein the megakaryocyte derivative includes MLC lysates, PLC, exosomes, megakaryocytes, or combinations thereof. Furthermore, methods for treating, repairing, or restoring conditions in subjects requiring treatment are provided herein, where megakaryocyte derivatives include fibroblast growth factor-2 (FGF-2); hepatocyte growth factor (HGF); insulin-like growth factor 1 (IGF-1); activated regulated normal T cell expression secretion factor (RANTES); neuronal growth factor (NGF); vascular endothelial growth factor (VEGF-A); vascular endothelial growth factor (VEGF-C); epidermal growth factor (EGF); transforming growth factor-β1 (TGF-β1); transforming growth factor-β2 (TGF-β1); and platelet-derived derivatives. The biomarker comprises one or more biomarkers selected from growth factor-AA (PDGF-AA); platelet-derived growth factor-BB (PDGF-BB); platelet-derived growth factor-AA / BB (PDGF-AA / BB); interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-12p40 (IL-12p40); interleukin-12p70 (IL-12p70); tissue metalloproteinase inhibitor 1 (TIMP-1); tissue metalloproteinase inhibitor 2 (TIMP-2); and tissue metalloproteinase inhibitor 3 (TIMP-3). Furthermore, methods for treating, repairing, or restoring a condition in an object requiring treatment of the condition are provided herein, where the condition is selected from dry eye disease, ocular surface disease, osteoarthritis, and wound healing. Furthermore, methods for treating, repairing, or restoring a condition in an object requiring treatment of the condition are provided herein, wherein the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, a neurotropic agent, an anti-hormone agent, or an immunomodulator, or a combination thereof.Furthermore, methods for treating, restoring, or restoring a condition in a subject requiring treatment are provided herein, wherein multiple doses are administered daily, weekly, bi-weekly, every three weeks, or monthly. Furthermore, methods for treating, restoring, or restoring a condition in a subject requiring treatment are provided herein, wherein the composition is administered by one of the following routes of administration selected from topical, transdermal, or systemic routes of administration. Furthermore, methods for treating, restoring, or restoring a condition in a subject requiring treatment are provided herein, wherein the composition is administered by one of the following routes of administration selected from subconjunctival, sub-Tenon, intravitreous, anterior chamber, intravenous, intra-arterial, intramuscular, subcutaneous, inhalation, rectal, oral, vaginal, intraperitoneal, intra-articular, intraglandular, topical, ear, or oral routes of administration. Furthermore, methods for treating, restoring, or restoring a condition in an object requiring treatment are provided herein, wherein (a) the condition is a dry eye disease, (b) the treatment is not for cancer, and (c) the route of administration is ophthalmoscopy, intraglandular, subconjunctival, topical, sub-Tenon's, intravitreous, or anterior chamber. Furthermore, methods for treating, restoring, or restoring a condition in an object requiring treatment are provided herein, wherein the composition is diluted to a physiological concentration in a carrier, the carrier comprising a diluent or excipient. Furthermore, methods for treating, restoring, or restoring a condition in an object requiring treatment are provided herein, wherein the carrier is plasma or plasma substitute or plasma light or physiological saline. Furthermore, methods for treating, restoring, or restoring a condition in an object requiring treatment are provided herein, wherein the megakaryocyte derivative is prepared in a fluid device or bioreactor. 【0190】 A method for producing megakaryocyte derivatives is provided herein, comprising: culturing a population of progenitor cells ex vivo for a period of time for the progenitor cells to differentiate into mature megakaryocytes; isolating a population of MLCs, PLCs, or their derivatives by separating them from megakaryocytes; enriching the MLCs, PLCs, or their derivatives; optionally lysing the MLCs, PLCs, or their derivatives; optionally mixing them with donor-derived PRP, wherein the megakaryocyte derivative comprises MLC lysates, PLCs, exosomes, megakaryocytes, or combinations thereof. Furthermore, a method for producing megakaryocyte derivatives is provided herein, wherein the progenitor cells are human induced pluripotent stem cells (iPSCs), hematopoietic stem cells, embryonic stem cells (ESCs), immortalized megakaryotic progenitor cells, CD34 + Umbilical cord blood stem cells (UCB cells), CD34 + Mobilized peripheral blood cells (MPB cells) or CD34 +One or more bone marrow cells are selected. Furthermore, compositions for treating dry eye disease in subjects are provided herein, and such compositions are prepared according to one of the methods described herein. In addition, methods for producing megakaryocyte derivatives are provided herein, where the megakaryocyte derivatives are fibroblast growth factor-2 (FGF-2); hepatocyte growth factor (HGF); insulin-like growth factor 1 (IGF-1); activated regulated normal T cell expression secretion factor (RANTES); neuronal growth factor (NGF); vascular endothelial growth factor (VEGF-A); vascular endothelial growth factor (VEGF-C); epidermal growth factor (EGF); transforming growth factor-β1 (TGF-β1); transforming growth factor-β2 (TGF-β1); platelet-derived growth factor-AA (PD). The biomarkers include one or more selected from GF-AA; platelet-derived growth factor-BB (PDGF-BB); platelet-derived growth factor-AA / BB (PDGF-AA / BB); interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-12p40 (IL-12p40); interleukin-12p70 (IL-12p70); tissue metalloproteinase inhibitor 1 (TIMP-1); tissue metalloproteinase inhibitor 2 (TIMP-2); and tissue metalloproteinase inhibitor 3 (TIMP-3). Furthermore, a method for producing megakaryocyte derivatives is provided herein, wherein the megakaryocyte derivatives include an EGF concentration per unit total protein that is higher than the mean EGF concentration per unit total protein measured in human platelets, platelet-rich plasma (PRP), or multi-growth factor plasma (PRGF), or an EGF concentration per unit total protein that is higher than the corresponding EGF concentration in the total protein concentration of PRGF. Furthermore, a method for producing megakaryocyte derivatives is provided herein, wherein the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, a neurotropic agent, an anti-hormone agent, or an immunomodulator, or a combination thereof. Furthermore, a method for producing megakaryocyte derivatives is provided herein, wherein the megakaryocyte derivative does not contain red blood cells or hemoglobin contents or white blood cells. Furthermore, a method for producing megakaryocyte derivatives is provided herein, wherein the composition further comprises extracellular vesicles (EVs).Furthermore, a method for producing megakaryocyte derivatives is provided herein, wherein the composition is formulated for application to a site of injury or tissue damage for therapeutic use. Furthermore, a method for producing megakaryocyte derivatives is provided herein, wherein the composition further comprises another therapeutic agent. Furthermore, a method for producing megakaryocyte derivatives is provided herein, wherein the megakaryocyte derivatives are produced in a fluid device or bioreactor. [Examples] 【0191】 (Example 1) DED study in a mouse DS model showing significant improvement in dry eye disease (DED) status. The in vivo effects of megakaryocyte derivatives (e.g., PLC, MLC lysate) were measured in subjects with dry eye disease. Briefly, to test PLC, a mouse dry stress (DS)-induced DED model was used in the first in vivo study, as shown in Figure 2A. The DS model uses sustained-release administration of scopolamine, an acetylcholine antagonist, in combination with low humidity environmental conditions to inhibit tear production and induce DED similar to human DED in mice. 【0192】 A group of 30 eight-week-old female C57BI / 6 mice was evaluated for DED symptoms starting on day 0, then scopolamine patches were applied, and the mice were placed in a controlled environment (e.g., 15 L / min, <25% humidity). The scopolamine patches were replaced every two days for two weeks. Three days after DED induction, the mice were re-evaluated for DED symptoms and subsequently treated with a topical administration of 5 μL / eye of either diluted Form E PLC (n=10, twice daily), a vehicle control (n=10, twice daily), or 2% cyclosporine A (CsA) ointment (Optimmune®) (n=10, three times daily). The vehicle was used as a negative control, and CsA as a positive control. Follow-up evaluations of DED symptoms were performed on days 7, 10, and 14. On day 14, the mice were sacrificed and their eyes were dissected for histological evaluation. Form E is a frozen medium consisting of PlasmaLyte A and 250 mM trehalose. Upon thawing Form E, it was diluted with sterile water to achieve a final osmotic pressure of 280–300 mOsm and a pH of 6.7–7. Optimmune® was used as prescribed. 【0193】 The DED status of these animals was assessed on days 0, 3, 7, 10, and 14 using corneal fluorescein staining and the phenol red thread test. Corneal fluorescein was used as the primary endpoint, and the results are shown in Figure 3. Here, corneal defects were examined by slit-lamp observation using blue light after dropping 0.5 μL of 0.5% sodium fluorescein. Defects were assessed by measuring the degree of damage on the corneal surface, as indicated by staining for punctate keratitis. The criteria are based on the National Eye Institute (NEI) grading system, which divides the cornea into five regions and grades the amount of staining in each region from 0 to 3 according to punctate fluorescein staining. The maximum score is 15. Table 1 provides the criteria for grading. [Table 1] 【0194】 Figure 2B presents the results of a study using PLC as a test treatment. CsA was used as a positive control. * indicates a statistically significant difference (p<0.05) compared to the vehicle, showing a significant difference in corneal damage reduction compared to the vehicle at days 7, 10, and 14, thus indicating the success of the PLC treatment. It should be noted that no significant difference was observed between PLC and CsA at any point in time. 【0195】 Further evaluation was achieved using a phenol red thread test, the results of which are provided in Figure 2C. Here, tear production was measured by a cotton thread test at the outer canthus of the conjunctival fornix for 30 seconds. Figure 2C shows the results of treatment with PLC. CsA was used as a positive control. Furthermore, * indicates a statistically significant difference (p<0.05) compared to the vehicle, and there was a significant difference in increased tear production compared to the vehicle on days 7 and 10, thus indicating the success of treatment with PLC. It should be noted again that there was no significant difference between PLC and CsA at any point in time. 【0196】 In a second follow-up study to test PLC, a mouse DS-induced DED model was used. The second study was conducted using 60 female mice. Dryness stress was induced in the same manner as in the first study and administered to the subjects twice daily from day 3 to day 14. Cyclosporine A was administered in the form of Restasis® eye drops. In addition to vehicle controls, an untreated negative control arm was also included. The test groups were administered PLC and two different doses of MLC lysate: MLC lysate match dose and MLC lysate maximum dose. The MLC lysate match dose contained the same total protein content as the PLC dose used in both studies, while the MLC lysate maximum dose contained approximately 7.3 times more total protein content than the PLC dose. Further study parameters are as follows: The subjects were divided into six groups, which included untreated (n=10), vehicle (n=10), PLC (n=10), MLC dissolved at matched dose (n=10), MLC dissolved at maximum dose (n=10), and Restasis® 0.05% emulsion (n=10). All formulations were prepared using Form E (PlasmaLyte, 150 mM trehalose). 【0197】 Figures 2D and 2E show the effect of the DS-induced DED model on goblet cells. Goblet cells in the right eye were counted total around the temporal, central, and nasal regions of the conjunctiva. Average cell count per mm of tissue from the eyelid to the conjunctiva (including the fornix) for all three sections per mouse. Mice in the vehicle cohort show a significant reduction in goblet cells compared to PLC-treated animals (Figure 2D). Without being bound by any particular theory, the analysis in Figure 2D suggests that the recovery of goblet cell loss may be the mechanism of action for the positive effect of PLC observed in a dryness stress model of ocular surface disease. Figure 2E shows representative histological results, with the central region shown by periodate-Schiff (PAS) staining. The analysis in Figure 2E suggests that fewer goblet cells are present in vehicle-treated animals compared to PLC-treated animals. 【0198】 Figure 3 shows the combined data from the first and second studies. Each eye was scored independently and compared to its own pre-treatment baseline (day 3). The vehicle and PLC arms were pooled from the two independent studies, while the other arms were performed only in the second study. Error bars = 95% confidence interval. Here, PRB v1 PLC refers to PLC produced using Method 1 described below, PRB v2 MLC lysate refers to MLC lysate produced using Method 2 described below, and PRB v3 MLC lysate refers to MLC lysate produced using Method 3 described below. Statistical analysis was performed at each time point using the Kruskal-Wallis rank-sum test, which revealed significant differences between groups at all three time points (p<0.0001). Post-hoc testing was performed using Dunn's multiple comparison test. Multiple-adjusted p-values ​​were calculated between each group at each time point. The test samples showed significantly improved CFS staining compared to untreated or vehicle-treated mice. Comparisons are indicated by asterisks: *p<0.05, **p<0.01, ***p<0.001. 【0199】 In summary, analysis of the results indicates that megakaryocyte derivatives (e.g., PLC, MLC lysates) can improve corneal fluorescein staining (CFS) in subjects with dry eye disease, possibly by restoring goblet cell loss. (Example 2) Model - TSP1 knockout mouse model of Sjögren's syndrome 【0200】 The effects of megakaryocyte derivatives (e.g., PLC, MLC lysate) were measured in subjects with Sjögren's syndrome. A TSP1 knockout mouse model of Sjögren's syndrome was used in in vivo studies as shown in Figure 4A to test PLC. Baseline corneal fluorescein staining (CFS) was performed on each eye in a cohort of 4-6 12-week-old male TSP1 knockout mice. Mice were treated with PLC or MLC lysate twice daily in both eyes for 14 days. PRGF was used as a positive control. The vehicle was used as a negative control. Following the final treatment, CFS was measured again in each eye and compared to the baseline CFS score on day 12. The four comparative studies in Study 2 were conducted as summarized below. Study 2.1 (n=14 mice, 28 eyes) included the following groups: vehicle (n=8), PLC (n=10), and MLC lysate (21100g lot 1) (n=10). Study 2.2A (n=11 mice, 22 eyes) included the following groups: vehicle (n=10) and MLC lysate (21100g lot 2) (n=12). Study 2.2B (n=9 mice, 18 eyes) included the following groups: vehicle (n=8) and PRGF (n=9). Study 2.3 (n=20 mice, 40 eyes) included the following groups: vehicle (n=10), 1.5x dose MLC lysate (200g lot 3) (n=10), 0.3x dose MLC lysate (200g lot 3) (n=10), and 0.06x dose MLC lysate (200g lot 3) (n=10). Study 2.3 included three doses of MLC lysate (1.5x, 0.3x, and 0.06x of total protein volume lot 3, similar to the PLC arms in Studies 2.1 and 2.2A), and the observation period was extended by an additional two weeks after the final dose to assess the duration of effect. 【0201】 Figure 4B shows the pooled results from Study 2. Analysis of Figure 4B shows that topical administration of various megakaryocyte derivatives can reverse the progression of dry eye disease symptoms in TSP1 knockout mice. The highest dose of MLC lysate tested (1.5 times the total protein amount, as with PLC, in Method 2) appeared to lose its effect, suggesting that such high concentrations may not be ideal and that the optimal dose of MLC lysate may be lower. "MLC lysate fast spin" represents the pooled data from Method 1. "MLC lysate slow spin" is from Method 2. One-way ANOVA: p<0.0001 (showing significant differences between means). Error bars = 95% confidence interval. Dunnett's multiple comparison test (vs. vehicle): ***p<0.001; **p<0.01; *p<0.05; ns are not significant. 【0202】 Figure 4C shows a representative image from Study 2.2A with marked areas of corneal fluorescein staining. Analysis of Figure 4C shows that locally applied MLC solution (twice daily for two weeks) significantly improves corneal barrier integrity compared to vehicle-treated corneas. 【0203】 Figure 4D shows a comparison of fluorescein scores in Study 2.3 as a function of time. In Study 2.3, an extension of the observation period by an additional two weeks after the final dose allowed for a preliminary assessment of the duration of the effect. CFS scoring was performed at 14 weeks of age (i.e., at the end of treatment) and again at 16 weeks of age (i.e., two weeks after discontinuation of treatment), and the score for each eye was compared to the baseline score measured at 12 weeks. The results show that treatment with megakaryocyte derivatives can improve corneal barrier integrity for at least two weeks after discontinuation of treatment. Error bars - 95% confidence interval. *p<0.05, **p<0.01 compared to vehicle (Mann-Whitney T-test). 【0204】 Based on the analysis of the results, it was shown that megakaryocyte derivatives (e.g., PLC, MLC lysate) can improve corneal fluorescein staining (CFS) in subjects with Sjögren's syndrome. Furthermore, it was shown that the positive effects of the treatment may persist even after discontinuation of treatment. Therefore, Sjögren's syndrome can be treated with megakaryocyte derivatives (e.g., PLC, MLC lysate) using a drug-free treatment protocol. (Example 3) Method for producing megakaryocyte derivatives 【0205】 Various methods for preparing megakaryocyte derivatives are described herein. 【0206】 Method 1: PLC was produced using the method described above. Briefly, the method was performed in a microfluidic bioreactor (BioR). Approximately 3.2e9 MLCs were thawed and centrifuged to remove DMSO. The MLCs were resuspended to a concentration of approximately 3.33e7 / ml in CO2-independent medium containing 2% Pluronics® F127. Subsequently, the MLCs were subjected to a spin-priming process by incubating 480 mL of resuspended MLC in a 0.5 lb PBS container at 35 rpm for 18 hours. The spin-primed MLCs were seeded into a 2 × 16 layer 3 μm pore platelet bioreactor. The system was run with 375 mL of medium to ensure complete collection of the product from the MLCs. The product was concentrated and diafiltration by tangential flow filtration (TFF). The product was diafiltration seven times in Plasmalyte, and then trehalose was added to 250 mM for cryopreservation. The product was dispensed into disposable vials for each treatment and stored at -80°C. Each treatment vial was thawed at room temperature (RT) immediately before use. 【0207】 Method 2: Using the same method, also referred to herein as MLC Lysates Lots 1 and 2, MLC lysates were produced by high-speed (21,100 g) centrifugation. Briefly, the MLC was thawed and centrifuged to remove DMSO. Subsequently, the MLC was resuspended in a mixture of Plasmalyte and water with 150 mM trehalose at either 5 e7 / mL (for standard doses, both Lots 1 and 2) or 2 e8 / mL (for maximum doses, Lot 1 only). Freeze-thaw thawing was performed by three cyclic incubations of the MLC at -80°C and RT. The MLC lysates were vortexed between each freeze-thaw cycle. The MLC lysates were clarified by high-speed (21,100 × g) centrifugation. The MLC lysates were collected as supernatant. BCA total protein analysis was performed to determine the total protein concentration of the MLC lysates, and the results were used to determine in vivo drug dosage. The MLC lysates were dispensed into disposable vials for each treatment and stored at -80°C. Each treatment vial was thawed using RT immediately before use. 【0208】 Method 3: Using the method also referred to herein as MLC Solvent Lot 3, MLC solubils were produced by processing with low-speed (200 g) centrifugation. Briefly, the MLC was thawed and centrifuged to remove DMSO. Subsequently, the MLC was resuspended in a mixture of Plasmalyte and water with 150 mM trehalose at either 5 e7 / mL (relative to standard dose) or 2 e8 / mL (relative to maximum dose). Freeze-thaw thawing was carried out by three cyclic incubations of the MLC at -80°C and RT. The MLC solubils were vortexed between each freeze-thaw cycle. Next, the MLC solubils were clarified by low-speed (200 × g) centrifugation. The clarified solubils were either left at full strength or partially diluted 5-fold or 25-fold with 150 mM trehalose in a mixture of Plasmalyte and water. Various undiluted and diluted MLC solutions were dispensed into disposable vials for each treatment and stored at -80°C. Each treatment vial was thawed by RT immediately before use. 【0209】 Method 4: Using the same method, MLC lysates were produced by processing through a 0.65 μm filter. Briefly, the MLC was thawed and centrifuged to remove DMSO. Subsequently, the MLC was resuspended in a mixture of plasmalyte and water at 5 e7 / mL with 150 mM trehalose. Freeze-thaw thawing was performed by three cyclic incubations of the MLC at -80°C and RT. The resulting product solution was clarified by vacuum filtration through a 20 μm uberstrainer. The resulting product solution was further clarified by passing it through a 0.65 μm polycarbonate track etch (PCTE) membrane in a filter cassette. Next, the filtered product was dispensed and stored at -80°C. 【0210】 Method 5: Using the same method, MLC lysates were produced by processing through a 5 μm filter. Briefly, the MLC was thawed and centrifuged to remove DMSO. Subsequently, the MLC was resuspended in water at 5e7 / mL. Freeze-thaw thawing was performed by three cyclic incubations of the MLC at -80°C and RT. The resulting product solution was clarified by passing it through a syringe filter cassette containing a 20 μm PCTE membrane. The resulting product solution was further purified by passing it through a 5 μm PCTE membrane in a second filter cassette. The final product was formulated to contain 150 mM trehalose, dispensed, and stored at -80°C. 【0211】 Method 6: Using the same method, MLC lysates were produced by processing through a 0.22 μm filter. Briefly, the MLC was thawed and centrifuged to remove DMSO. Subsequently, the MLC was resuspended in water at 5 e7 / mL. Freeze-thaw thawing was performed by three cyclic incubations of the MLC at -80°C and RT. The obtained MLC lysate was clarified by passing it through a syringe filter cassette containing a 20 μm PCTE membrane. The MLC lysate was further clarified by passing it through a 5 μm PCTE membrane in a second filter cassette. As a final step, the obtained product solution was passed through a 0.22 μm PES filter. The final product was formulated to contain 150 mM trehalose, dispensed, and stored at -80°C. (Example 4) Method for producing multi-growth factor platelets (PRGF) 【0212】 A method for producing multi-growth factor platelets (PRGF) is described herein. Briefly, donor blood was collected, used to isolate platelet-rich plasma (PRP), and further processed according to approved guidance and protocols to produce PRGF. The donated whole blood was first centrifuged at 150 × g to allow for the collection of plasma from red blood cells and the buffy coat. Prostaglandin E1 (PGE1) was added to the plasma sample, and the platelets were concentrated by centrifugation at 460 × g. The platelet-poor fraction (upper two-thirds) was removed and discarded after centrifugation. The pellet was resuspended using the remaining lower one-third of the plasma. The resulting PRP was activated by adding 228 mM CaCl2 and incubating at 37°C for at least 15 minutes, or until thrombus formation was observed. The activated PRP was centrifuged at 16600 × g. The supernatant was isolated and diluted to 20% (v / v) with physiological saline. The final product was dispensed into disposable vials and stored at -80°C. The disposable vials were thawed by RT immediately before use. (Example 5) Profiling of growth factors, cytokines, and other related proteins in MLCs and MLC derivatives 【0213】 Profiling for growth factors, cytokines, and other related proteins was determined for MLC, crude MLC lysate (i.e., no further processing), PLC, and MLC lysate processed in different ways. PLC was prepared using Method 1 as described in Example 3. MLC was lysed and processed using Methods 2, 3, 4, and 5 as described in Example 3. "Homemade" PRGF (multi-growth factor plasma) was prepared using the method described in Example 4, while "Endoret-PRGF" was obtained from a manufacturer that prepared it from human blood using the Endoret eye drop kit. For preparation for biomarker profiling, each sample was incubated with RIPA Lysis Buffer (10 × RIPA, 100 × PI), followed by three cycles of 1 minute of maximum force vortexing and 10 minutes of incubation on ice, and then clarified by high-speed centrifugation. The total protein concentration of all clarified lysates was measured using the Pierce BCA Assay. Next, the samples were diluted with Plasmalyte-A to a final concentration of 1 mg / mL total protein for analysis. Further dilutions of up to 500-fold were applied to the samples to accommodate the range of the human panel assay. Growth factor and cytokine profiling was used to simultaneously analyze multiple biomarkers (over 70 cytokines, GF, and metalloproteinases). PRGF (multi-growth factor plasma, PRP excipient) was used as a control. Figure 5 is a heatmap display of the profiled cytokines and growth factors, showing the relative concentrations of cytokines and growth factors as measured by profiling. The presented data represent the Z-score of the full intensity concentration of growth factors and cytokines per 1 volume of sample. Figures 6A-6D and 7A-7D show the actual concentrations of selected factors, indicating that various types of MLC contain high concentrations of desirable factors (epitheliotropic and platelet-related factors, TIMPS which inhibits MMP histolysis, and interleukins which polarize T cells and secrete dissipation-promoting cytokines) compared to human blood-derived PRGF.Therefore, MLC and its lysates are distinct products of PRGF. 【0214】 In summary, the analysis of the results indicates that MLC and its derivatives can offer unique therapeutic benefits compared to PRGF. (Example 6) Biomarker profiling of megakaryocyte derivatives using enzyme-linked immunosorbent assay (ELISA) 【0215】 Biomarker profiling was performed on megakaryocyte derivatives (e.g., MLC lysates and PLCs) using enzyme-linked immunosorbent assay (ELISA) for the samples prepared in Examples 5 and 6. Briefly, the total protein concentration for each sample was determined using total protein analysis (BCA). Figure 8A shows the BCA results for all samples tested. The results indicate that megakaryocyte derivatives contain different overall protein levels depending on how they were produced / processed. * indicates a representative set of samples produced using the same process as in the in vivo study. 【0216】 Figures 8B and 8C provide ELISA-based quantifications of EGF and PDGF-BB in various megakaryocyte derivatives, respectively. Analysis in Figure 8B shows that the EGF profiling of the samples exhibits a similar trend to that observed in Examples 5 and 6. * indicates representative samples produced and processed using the same process as Method 2 (21100g) and Method 3 (200g). The gray bars represent Method 3 (200g). 【0217】 Analysis of ELISA results further confirmed that MLC lysates and PLC contain biomarker profiles specific to PRGF. Therefore, MLC lysates and PLC may offer specific therapeutic benefits for PRGF. (Example 7) Particle size analysis of megakaryocyte derivatives 【0218】 Particle size analysis was performed on various megakaryocyte derivatives (e.g., MLC lysates and PLC). Briefly, the particle size distribution was measured using an nCS1 particle analyzer (Spectradyne, Signal Hill, CA) on the supernatant of crude MLC lysates filtered through PLC and a 20 μm PCTE / 5 μm PCTE or 20 μm PCTE / 5 μm PCTE / 0.22 μm PES membrane, and MLC lysates centrifuged at 200 g or 21,100 g. Samples were diluted 5 to 200 times in a diluent filtered through 0.22 μm (1 × PBS with 1% Tween®-20). Subsequently, the diluted samples were loaded into a C-10k nCS1 analysis cartridge. Data were obtained for N > 6000 unless otherwise noted. The raw data were then processed using Viewer® software (Spectradyne). Figure 9A shows the particle size distribution results for the five samples tested. Figure 9B shows the particle concentrations (1750–10000 nm) in each sample, represented as a bar graph. Virtually all micro-sized particles are absent in the megakaryocyte derivatives centrifuged at 21100 g and filtered at 0.22 μm. This indicates that, depending on the processing steps, megakaryocyte derivatives can be naturally produced that are primarily microparticles (PLC), naturally primarily soluble (21100 g supernatant and filtered at 0.2 μm), or a combination of both (200 g supernatant and filtered at 5 μm). Therefore, the preparation of megakaryocyte derivatives can be tailored to suit the desired properties. (Example 8) Effects of megakaryocyte derivatives on human corneal epithelial (HCE-T) cell proliferation 【0219】 The effect of MLC lysate on the proliferation of human corneal epithelial (HCE-T) cells was measured. Briefly, HCE-T cells were seeded in Minimal KGM-Gold medium (Lonza, Walkersville, MD) at a rate of 2,500 cells per well on E-Plate 96 (Agilent Technologies, Santa Clara, CA). Minimal KGM-Gold medium contains all KGM-Gold BulletKit components except EGF (positive control), MLC lysate, and / or bovine pituitary extract. Phosphate-buffered water (PBS) was used for the negative control vehicle only. After 24 hours, cells were treated with indicated concentrations of MLC lysate, EGF (SinoBiological Inc., Beijing, China), or PBS. Subsequently, the E-plates were returned to the xCELLigence RTCA instrument for a further 48 hours for data collection. The area under the curve (AUC) of the normalized cell index was calculated using RTCA Software Pro and then normalized for the vehicle. Figure 10A shows the results of a 72-hour time course study of the effect of EGF on HCE-T cell proliferation at concentrations ranging from 30.4 pg / ml to 31.2 ng / ml. Figure 10B is an alternative representation of Figure 10A, showing the normalized area under the curve for the normalized cell index for each concentration. The analysis in Figures 10A and 10B demonstrates that EGF promotes corneal epithelial cell proliferation in a dose-dependent manner. Figure 11A shows the results of a 72-hour time course study of the effect of MLC lysates treated by high-speed centrifugation on HCE-T cell proliferation at doses ranging from 0.025% (v / v) to 0.75% (v / v). Figure 11B is an alternative representation of Figure 11A, showing the normalized area under the curve for the normalized cell index for each concentration. Figure 11C shows the results of a 72-hour time-course study of the effect of MLC lysates treated with 5 μm filtration followed by 0.22 μm filtration on the proliferation of HCE-T cells at doses ranging from 0.0128% (v / v) to 0.1% (v / v). Figure 11D is an alternative representation of Figure 11C, showing the normalized area under the curve for the normalized cell index for each concentration.Analysis in Figures 11A-11D shows that HCE-T cell proliferation can be enhanced by the addition of MLC lysates. This biological activity has been verified, demonstrating a potential mechanism of action that contributes to the alleviation of ocular surface diseases. 【0220】 To further confirm the proliferative effect of megakaryocyte derivatives on HCE-T cells, an orthogonal assay, a carboxyfluorescein diacetate succinmidyl ester (CFSE)-based flow cytometry assay, was performed. Briefly, HCE-T cells were labeled with CFSE (Invitrogen Cell Trace®), seeded at 10,000 cells per well, and grown in complete medium (KGM-Gold, Lonza). The medium was changed to basal medium (KBM-Gold, Lonza), and MLC lysates produced using a 5 μm filter (5 μm 0.5%~2%) and centrifugation at 21,100 × g (21k 2%~4%), or the corresponding vehicle, were added to the cells and incubated for a further 60–72 hours. Assay controls included complete medium, basal medium, and PRGF, all incubated for the same period as the test samples. A Miltenyi MACSquant flow cytometer was used to record the geometric mean fluorescence intensity (MFI) for CFSE-labeled cells. Figure 12A shows the MFI observed for each test condition, where proliferation is inversely correlated with the MFI value. Figure 12B is an alternative representation of Figure 12A, showing the normalized and inverted MFI relative to the KBM control, where a direct correlation is shown between increased proliferation and the normalized MFI value. Samples with values ​​between 1 and 1.3 (dotted line) do not show increased proliferation. Samples above 1.3 (dotted line) indicate increased proliferation compared to the negative control for each test condition. Analysis of Figures 12A and 12B demonstrates that HCE-T cell proliferation can be enhanced by treatment with megakaryocyte derivatives, verifying their bioactivity and demonstrating a potential mechanism of action that contributes to the alleviation of ocular surface disease symptoms. 【0221】 In summary, analysis of the results indicates that the specific biomarker composition of MLC lysates is favorably useful in alleviating symptoms of ocular surface diseases by promoting the proliferation of HCE-T cells. (Example 9) Osteoarthritis model for in vivo efficacy studies 【0222】 The in vivo effects of MLC lysates and PLC were measured in subjects with osteoarthritis. A rat medial meniscal tear (MMT) model with osteoarthritis (OA) was used in an in vivo study to test the efficacy of megakaryocyte derivatives. Figure 13A shows a schematic of the study design. As shown in Figure 13A, Lewis rats underwent meniscal tear surgery on day 0. Dynamic weight loading and electron von Frey filament analysis were performed at 7, 14, 21, and 28 days post-surgery. Subjects were administered MLC lysates or PLC by intra-articular injection after DWB and EVFF analysis at 7, 14, and 21 days post-surgery. Serum was collected on day 28 post-surgery, and the animals were sacrificed to allow for joint histological examination. PRP and recombinant human FGF-18 were used as positive controls. For negative controls, only the vehicle was used. In summary, the study included the following groups: no surgery (n=5), MMT surgery + vehicle (n=15), MMT surgery + platelet-like cells (n=15), MMT surgery + platelet-rich plasma (n=15), MMT surgery + rFGF-18 (n=15), and MMT surgery + MLC lysate (n=15). 【0223】 Figures 13B and 13C show the efficacy results of MLC lysate and PLC in a rat medial meniscus tear model of osteoarthritis using dynamic weight-bearing tests and electron von Frey filament tests, respectively. As shown in Figure 13B, MLC lysate, PLC, and PRP showed improved pain profiles compared to recombinant human FGF-18, which showed a gradually increasing signal over time in the dynamic weight-bearing test compared to the vehicle. As shown in Figure 13C, in electron von Frey filament analysis, PLC and MLC lysate treatment yielded thresholds comparable to vehicle treatment, while recombinant human FGF-18 showed a significant decrease in threshold and higher pain sensitivity. 【0224】 As outlined in Figure 13A, histological analysis of the tissues under test was performed 28 days after surgery. Figure 13D shows representative H&E stained histological images of joints treated with vehicle and PLC. Degeneration of articular cartilage was observed in joints treated with vehicle (left), while the articular cartilage layer was intact in joints treated with PLC (right). Figure 13E shows a quantitative representation of cartilage degeneration observed in the histological analysis of each subject. As shown in Figure 13E, a statistically significant reduction in the width of substantial cartilage degeneration was observed when treated with PLC compared to vehicle (p < 0.05). Treatment with recombinant human FGF-18 also showed a significant reduction when compared to vehicle treatment (p < 0.001). Furthermore, synovitis score and medial tibial spine measurement were performed based on the analysis of histological data. Figure 13F shows the evaluation of synovitis in the joints of the treated subjects. Analysis of Figure 13F showed that PLC, PRP, and MLC lysates did not show a clear difference compared to vehicle treatment, while recombinant human FGF-18 showed a significant increase in the synovitis score (**** = p,0.0001). Figure 13G shows the results of quantification of bone spurs in the treated joints of the subjects. Analysis of Figure 13G showed that PLC, PRP, and MLC lysates did not show a clear difference in medial tibial spine measurement compared to vehicle treatment, while treatment with recombinant human FGF-18 resulted in a significant increase (**** = p < 0.0001). Naive mice were observed to have a significant decrease in medial tibial spine measurement compared to vehicle-treated mice (**** = p < 0.0001). 【0225】 In summary, analysis of the results has shown that the unique biomarker composition of PLC serves suitably for the preservation of intact articular cartilage and / or the improvement / reversal of cartilage degenerative conditions in osteoarthritis subjects. (Example 10) Effect of PLC on human umbilical vein endothelial cell (HUVEC) migration 【0226】 The in vivo effect of PLC on the migration of human umbilical vein endothelial cells (HUVEC) was measured. Briefly, a rat medial meniscus scratch assay was performed to evaluate the effect of PLC on HUVEC migration. HUVEC (10,000 cells per well) were seeded into E-Plate Wound 96 (Agilent Technologies, Santa Clara, CA) in complete EGM-2 medium (Lonza, Walkersville, MD) and incubated for 24 hours in an xCELLigence Real-Time Cell Analysis instrument (Agilent Technologies). Subsequently, a scratch was generated across each well using an AccuWound 96 Scratch Tool (Agilent Technologies). The wells were rinsed with PBS and replaced with minimal medium (EGM-2 without VEGF, EGF, or IGF and containing 0.1% FBS and 1 ng / mL of bFGF). An E-plate insert (Agilent Technologies) was placed into the wells, and the following treatments were applied to the upper chamber of the insert: vehicle, recombinant VEGF (100 ng / mL), or PLC*. Recombinant VEGF was used as a positive control. The PLC sample was produced using the same process as described in Method 1 of Example 3. Next, the E-plate was returned to the xCELLigence RTCA instrument for an additional 96 hours of data collection. Figure 14A shows the time-course data (120 hours) of the obtained cell index, and Figure 14B shows the AUC data normalized to the vehicle. This data shows that PLC can promote HUVEC migration in the scratch assay, verifying the bioactivity of megakaryocyte derivatives and demonstrating a potential mechanism of action contributing to wound healing. 【0227】 In summary, analysis of the results has shown that the specific biomarker composition of PLC is suitably useful for promoting wound healing in a subject by promoting the migration of HUVEC. (Example 11) In vivo efficacy of megakaryocyte derivatives in wound healing 【0228】 The in vivo effect of PLC on wound healing in diabetic subjects was measured. Briefly, a db / db diabetic mouse model was used in an in vivo study to evaluate the efficacy of megakaryocyte derivatives (PLC and MLC lysates) in wound healing. Figure 15A shows an overview of the study design. As shown in Figure 15A, glucose measurements were performed on day 0 to ensure that the mice were in a diabetic state. Subsequently, the mice underwent surgery, creating two identical excision wounds on their backs via biopsy punches. Immediately after surgery and for the following 3 days, 50 μL of PLC was administered topically twice daily for 3 days. PRP was used as a positive control. For the negative control, only the vehicle was used. Wound images were captured using digital imaging on the day of surgery and at 3, 6, 8, and 10 days after surgery. Animals were sacrificed 10 days after in-life measurements, and the wounds were excised for histological analysis. 【0229】 Digital imaging and subsequent wound area measurements were performed over a 10-day period after treatment of the test samples. Figure 15B shows the wound closure kinetics for subjects treated with each test sample. Analysis of Figure 15B shows that treatment with PLC and PRP resulted in faster wound closure kinetics on days 3 and 6 compared to vehicle treatment (*=p<0.05). 【0230】 Figure 15C shows the histological results of diabetic mouse wounds. Representative histological sections of wounds stained with H&E (two representative histological images per condition) highlight treatment-related differences in wound healing, including granulation tissue formation. Both PLC and PRP treatments result in improved granulation tissue area compared to vehicle treatment. 【0231】 Figures 15D–15F provide a quantitative analysis of wound closure (Figure 15D), granulation tissue area (Figure 15E), and epidermal tissue area (Figure 15F) based on the analysis of wound histological images. A significant difference was observed in granulation tissue area of ​​wounds treated with PLC and PRP compared to the vehicle (****=p<0.0001). PLC and PRP treatments show a tendency towards increased wound closure and epidermal tissue area. 【0232】 Tissue sections were also treated with fluorescent endomucin antibody as a substitute for blood vessels within healing wounds. Figure 15G shows endomucin-based histological visualizations of the vascular system for subjects treated with vehicle, PLC (center), and PRP (right). Figure 15H shows quantification of endomucin fluorescence signaling, which shows that PLC and PRP have a statistically significant increase in endomucin compared to vehicle treatment (*=p<0.05). 【0233】 In summary, analysis of the results indicates that the specific biomarker composition of PLC is suitably beneficial for wound healing in diabetic patients. Wound healing may be associated with increased endomucin levels in PLC-treated subjects. (Example 12) Fabrication of MLC 【0234】 A method for producing MLCs for use in any of the embodiments described herein is described herein. Briefly, Figure 16A shows a schematic diagram of the differentiation process. A culture medium containing the specified components was used at each stage to induce cell differentiation into megakaryocyte-like cells (MLCs). One vial of human induced pluripotent stem cell working cell bank (hiPSC WCB) can be grown to create multiple vials of cryopreserved intermediate (CPI) cells. First, the CPI vials were thawed to initiate the differentiation process. Subsequently, after 2D proliferation of hiPSCs from the CPI vials for approximately 3 days, the cells were harvested and transferred to a vertical rotating device, such as a vertical wheel bioreactor, to allow 3D aggregation of hiPSCs from single-cell suspension for approximately 1 day. In Stage 1, the aggregates were transferred to a spinner flask and differentiation into hematopoietic endothelium was initiated over approximately 6 days. In Stage 2, MLC precursor cells (pre-MLCs) were released from the aggregated hematopoietic endothelium and harvested daily into separate containers for Stage 2.5 proliferation over approximately 4 days. Stage 3 was the final maturation process from pre-MLC to MLC, and its duration was approximately 3 days. The MLC was then harvested for cryopreservation and / or megakaryocyte derivative production. Freshly harvested MLC can be used to optimize processing time and efficiency. 【0235】 Figure 16B shows further details regarding stages 2.5 and 3 (enlarged images of the differentiation process in stages 2.5 and 3 as shown in Figure 16A). In particular, Figure 16B describes the harvesting strategy for batch pre-MLC to MLC maturation culture. Stages 2 through 2.5 are where preMLCs are released from hematopoietic endothelial aggregates as needed. The released cells were collected daily from spinner flasks and transferred to G-Rex containers for further proliferation and maturation. All G-Rex containers were harvested as a single batch at the end of the process. (Example 13) PLC manufacturing 【0236】 A method for preparing PLC for use in any of the embodiments described herein is described herein. Briefly, Figure 17A shows a schematic diagram of the PLC production process. MLCs were thawed, centrifuged, DMSO removed, and the cells washed. Subsequently, the MLCs were resuspended in BioR medium. The MLC suspension was spin-primed in a "PBS" spinner. The MLCs were processed in a biomimetic microfluidic platelet bioreactor (BioR). The BioR product was collected and processed by approximately 300-400-fold concentration of TFF and diafiltration with Plasmalyte A. The TFF product was analyzed for OD600 and formulated to a target dose of 70 FAU / mL. The DP was aliquoted, frozen, and stored at -80°C. 【0237】 Figure 17B shows an exemplary microfluidic bioreactor (BioR) device for processing MLCs. The BioR device contained a PCTE (polycarbonate track etch) membrane with 3 μm pores. MLCs were seeded onto the membrane, a pressure gradient was generated, and the MLCs were stimulated to produce PLC. 【0238】 From the foregoing description, it is clear that the embodiments of this disclosure may be modified and adapted to various uses and conditions. Such embodiments are also within the scope of the following claims. Any description of a list of elements in any definition of a variable herein includes the definition of that variable as any single element or as a combination (or partial combination) of the enumerated elements. Any description of an embodiment herein includes that embodiment as any single embodiment or as a combination with any other embodiment or a part thereof. 【0239】 All patents and publications referenced herein are incorporated herein by reference to the same extent that separate patents and publications are specifically and individually indicated to be incorporated by reference, respectively.

Claims

[Claim 1] A method for treating, repairing, or restoring a condition in a subject requiring such treatment, comprising administering to the subject an effective amount of a composition comprising a megakaryocyte derivative, wherein the condition is a dry eye disease, the route of administration is by eye drops or intraglandular instillation, and the megakaryocyte derivative comprises megakaryocyte-like cell (MLC) lysate, platelet-like cell (PLC), or a combination thereof. [Claim 2] The method according to claim 1, further comprising a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, a neurotropic agent, an anti-hormone agent, or an immunomodulator, or a combination thereof. [Claim 3] The method according to claim 2, wherein the tissue regeneration agent is (a) a growth factor selected from one or more of the following: transforming growth factor (TGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet-derived endothelial growth factor (PDEGF), platelet-derived angiogenic factor (PDAF), platelet factor 4 (PF-4), hepatocyte growth factor (HGF), or a combination thereof; and (b) one or more of the following: cytokines selected from one or more of the following: IL-1B, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17A, IL-23, TNF-alpha, or a combination thereof. [Claim 4] The megakaryocyte derivatives include fibroblast growth factor-2 (FGF-2); hepatocyte growth factor (HGF); insulin-like growth factor 1 (IGF-1); activated regulated normal T cell expression secretion factor (RANTES); neurotrophic growth factor (NGF); vascular endothelial growth factor (VEGF-A); vascular endothelial growth factor (VEGF-C); epidermal growth factor (EGF); transforming growth factor-β1 (TGF-β1); transforming growth factor-β2 (TGF-β1); platelet-derived growth factor-AA (PDGF-AA); platelet-derived growth factor-BB (PDG The method according to claim 1 or 2, comprising one or more biomarkers selected from F-BB; platelet-derived growth factor-AA / BB (PDGF-AA / BB); interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-12p40 (IL-12p40); interleukin-12p70 (IL-12p70); tissue metalloproteinase inhibitor 1 (TIMP-1); tissue metalloproteinase inhibitor 2 (TIMP-2); and tissue metalloproteinase inhibitor 3 (TIMP-3). [Claim 5] The method according to claim 4, wherein the megakaryocyte derivative contains an EGF concentration per unit total protein that is higher than the average EGF concentration per unit total protein measured in human platelets, platelet-rich plasma (PRP), or multi-growth factor plasma (PRGF). [Claim 6] The method according to any one of claims 1 to 5, wherein the megakaryocyte derivative is produced from cells derived from somatic cells, progenitor cells, or stem cell reprogramming and / or differentiation. [Claim 7] The method according to any one of claims 1 to 6, wherein the megakaryocyte derivative is not a cancer cell. [Claim 8] The method according to any one of claims 1 to 7, wherein the megakaryocyte derivative does not exhibit uncontrolled proliferation or tumorigenesis in vivo. [Claim 9] The method according to any one of claims 1 to 8, wherein the method reduces the injury recovery time. [Claim 10] The method according to any one of claims 1 to 9, wherein the method improves one or more signs and / or symptoms of a dry eye disease. [Claim 11] The method according to any one of claims 1 to 10, wherein the composition further comprises a lysate, serum, plasma, multi-growth factor plasma (PRGF), platelet-rich plasma (PRP), or any other blood derivative derived from the subject. [Claim 12] The method according to any one of claims 1 to 11, wherein the route of administration of the eye drops is subconjunctival or local. [Claim 13] The method according to claim 12, wherein the route of administration of the eye drops is under Tenon's. [Claim 14] The method according to claim 12, wherein the route of administration of the eye drops is intravitreous or intrachorium. [Claim 15] The method according to any one of claims 1 to 14, wherein the dry eye disorder is caused by Sjögren's syndrome or non-Sjögren's syndrome. [Claim 16] The method according to any one of claims 1 to 15, wherein the megakaryocyte derivative does not contain red blood cells or hemoglobin contents or white blood cells. [Claim 17] The method according to any one of claims 1 to 16, wherein the composition further comprises an extracellular vesicle (EV). [Claim 18] The method according to any one of claims 1 to 17, wherein the composition is formulated for application to a site of injury or tissue damage for therapeutic use. [Claim 19] The method according to claim 18, wherein the composition is formulated in a buffer, a diluent, or an excipient, or a combination thereof. [Claim 20] The method according to any one of claims 1 to 19, wherein the composition comprises a) 0.01 to 100% by weight of the megakaryocyte derivative, b) 0 to 90% by weight of a volume extender, and / or c) 0 to 90% by weight of at least one excipient or carrier, and optionally d) further comprising platelet-rich plasma (PRP), PRGF, plasma, serum, or other blood derivatives derived from the subject. [Claim 21] The method according to any one of claims 1 to 20, wherein the composition is freeze-dried. [Claim 22] The method according to any one of claims 1 to 20, wherein the composition is embedded in an implantable device. [Claim 23] The method according to any one of claims 1 to 20, wherein the composition is stored by freezing. [Claim 24] The method according to any one of claims 1 to 20, wherein the composition is administered topically to one or more sites of injury or disease or its vicinity. [Claim 25] The method according to any one of claims 1 to 24, wherein the composition further comprises another therapeutic agent. [Claim 26] A method for treating, repairing, or restoring a condition in a subject requiring such treatment, comprising administering to the subject multiple doses of an effective amount of a composition containing a megakaryocyte derivative, The aforementioned condition is selected from dry eye disease, ocular surface disease, osteoarthritis, and wound healing. The megakaryocyte derivative comprises MLC lysate, PLC, exosomes, megakaryocytes, or a combination thereof. The megakaryocyte derivatives include fibroblast growth factor-2 (FGF-2); hepatocyte growth factor (HGF); insulin-like growth factor 1 (IGF-1); activated regulated normal T cell expression secretion factor (RANTES); neuronal growth factor (NGF); vascular endothelial growth factor (VEGF-A); vascular endothelial growth factor (VEGF-C); epidermal growth factor (EGF); transforming growth factor-β1 (TGF-β1); transforming growth factor-β2 (TGF-β1); platelet-derived growth factor-AA (PDGF-AA); platelet-derived growth factor- A method comprising one or more biomarkers selected from BB (PDGF-BB); platelet-derived growth factor-AA / BB (PDGF-AA / BB); interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-12p40 (IL-12p40); interleukin-12p70 (IL-12p70); tissue metalloproteinase inhibitor 1 (TIMP-1); tissue metalloproteinase inhibitor 2 (TIMP-2); and tissue metalloproteinase inhibitor 3 (TIMP-3). [Claim 27] The method according to claim 26, further comprising a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, a neurotropic agent, an anti-hormone agent, or an immunomodulator, or a combination thereof. [Claim 28] The method according to claim 26 or 27, wherein the plurality of doses are administered daily, weekly, every other week, every three weeks, or monthly. [Claim 29] The method according to any one of claims 26 to 28, wherein the composition is administered by one of the following routes of administration selected from local, transdermal, or systemic routes. [Claim 30] The method according to any one of claims 26 to 28, wherein the composition is administered by one of the following routes of administration selected from subconjunctival, subtenon, intravitreous, anterior chamber, intravenous, intraarterial, intramuscular, subcutaneous, inhalation, rectal, oral, vaginal, abdominal, intraarticular, intraglandular, lacrimal gland, topical, ear, or oral administration routes. [Claim 31] The aforementioned condition is a dry eye disorder. The aforementioned treatment is not for cancer, The method according to any one of claims 26 to 28, wherein the route of administration is by eye instillation, intraglandular, subconjunctival, topical, sub-Tenon's, intravitreous, or anterior chamber. [Claim 32] The method according to any one of claims 26 to 30, wherein the composition is diluted to a physiological concentration in a carrier, and the carrier comprises a diluent or an excipient. [Claim 33] The method according to claim 32, wherein the carrier is plasma, plasma substitute, plasma light, or physiological saline. [Claim 34] A method for producing megakaryocyte derivatives, The process involves culturing a population of progenitor cells in ex vivo for a period of time during which the progenitor cells are differentiated into mature megakaryocytes, By separating from the megakaryocytes, a group of MLCs, PLCs, or their derivatives can be isolated. Concentrating the aforementioned MLC, PLC, or derivatives thereof, If necessary, dissolve the MLC, PLC, or their derivatives, If necessary, mix with PRP derived from the donor. Includes, A method wherein the megakaryocyte derivative comprises an MLC lysate, a PLC, an exosome, a megakaryocyte, or a combination thereof. [Claim 35] The aforementioned progenitor cells include human induced pluripotent stem cells (iPSCs), hematopoietic stem cells, embryonic stem cells (ESCs), immortalized megakaryocyte progenitor cells, and CD34. ＋ Umbilical cord blood stem cells (UCB cells), CD34 ＋ Mobilized peripheral blood cells (MPB cells) or CD34 ＋ The method according to claim 34, wherein one or more bone marrow cells are selected. [Claim 36] A composition for treating dry eye disease in a subject, prepared according to the method described in any one of claims 34 to 35. [Claim 37] The megakaryocyte derivatives include fibroblast growth factor-2 (FGF-2); hepatocyte growth factor (HGF); insulin-like growth factor 1 (IGF-1); activated regulated normal T cell expression secretion factor (RANTES); neuronal growth factor (NGF); vascular endothelial growth factor (VEGF-A); vascular endothelial growth factor (VEGF-C); epidermal growth factor (EGF); transforming growth factor-β1 (TGF-β1); transforming growth factor-β2 (TGF-β1); platelet-derived growth factor-AA (PDGF-AA); platelet-derived growth factor-BB (PDGF-AA). The composition according to claim 36, comprising one or more biomarkers selected from GF-BB; platelet-derived growth factor-AA / BB (PDGF-AA / BB); interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-12p40 (IL-12p40); interleukin-12p70 (IL-12p70); tissue metalloproteinase inhibitor 1 (TIMP-1); tissue metalloproteinase inhibitor 2 (TIMP-2); and tissue metalloproteinase inhibitor 3 (TIMP-3). [Claim 38] The composition according to claim 37, wherein the megakaryocyte derivative contains an EGF concentration per unit total protein that is higher than the average EGF concentration per unit total protein measured in human platelets, platelet-rich plasma (PRP), or multi-growth factor plasma (PRGF). [Claim 39] The composition according to any one of claims 36 to 38, further comprising a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, a neurotropic agent, an anti-hormone agent or an immunomodulator or a combination thereof. [Claim 40] The composition according to any one of claims 36 to 39, wherein the megakaryocyte derivative does not contain red blood cells or hemoglobin contents or white blood cells. [Claim 41] The composition according to any one of claims 36 to 40, further comprising extracellular vesicles (EVs). [Claim 42] A composition according to any one of claims 36 to 41, formulated for application to a site of injury or tissue damage for therapeutic use. [Claim 43] The composition according to any one of claims 36 to 42, further comprising another therapeutic agent. [Claim 44] The method according to claims 1, 28, and 34, wherein the megakaryocyte derivative is produced in a fluid device or bioreactor.

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