Anucleated cells as a source for the treatment of platelet-rich plasma-dependent disorders

Abstract

To provide novel anucleated cells as a source for treatment of platelet-rich plasma-dependent disorders.SOLUTION: Described herein are methods of using platelet-like cells or a variant thereof (PLC) or a derivative thereof or a lysate thereof, or platelet rich plasma (PRP) derived therefrom, to treat, repair or ameliorate diseases, disorders or injuries associated with dry eye, osteoarthritis, tendons, ligaments, bone repair, wound healing or wound healing related disorders, alopecia, or to rejuvenate or regenerate skin. Also described herein are methods of producing platelet rich plasma (PRP) from PLC or a derivative thereof or a lysate thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 024,587, filed May 14, 2020; U.S. Provisional Patent Application No. 63 / 106,009, filed October 27, 2020; and U.S. Provisional Patent Application No. 63 / 144,033, filed February 1, 2021, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] background Platelet-rich plasma (PRP) has been proposed to play a role in the medical treatment of wound healing or rapid repair of damaged tissues (such as cartilage, tendons, ligaments, and bones) or in the treatment of dry eye syndrome and other diseases. However, due to limited sample sizes of reported data from platelet donors, conclusive data regarding the beneficial effects of PRP in clinical procedures is lacking. Furthermore, every platelet donor is different, and therefore, the quality of PRP produced for medical procedures is inconsistent. Because every patient provides their own platelets as a source of PRP, currently used PRP suffers from several other drawbacks, such as batch-to-batch variability. Other drawbacks include the short lifespan of donor platelets, impurities or contaminants, or unavailability or shortage of supply. As a result of the lack of standardization of PRP products, the clinical effectiveness of PRP remains under debate. Therefore, there is an urgent need in the art for a safe, standardized, defined, and readily available product that can be developed and approved by regulatory agencies for human use, replacing the need to generate individual, unreliable PRP preparations from each patient, which raises doubts about its potential efficacy and use.

[0003] Osteoarthritis (OA) is an example of a disease with a significant unmet medical need, for which a treatment is urgently needed. Current OA treatment involves administering an NSAID and duloxetine to patients. If symptoms do not improve, oral duloxetine is initiated. If symptoms improve after three months, oral duloxetine treatment may be continued without pharmacological management and the progression of symptoms monitored, potentially extending the treatment course. If symptoms persist or progress after three months, patients needing short-term pain relief should receive intra-articular glucocorticoid injections. If symptoms continue to progress, the patient is deemed to require surgery and referred to an orthopedic surgeon. Therefore, new, less invasive, and better treatment options are needed for the treatment of OA and other diseases with unmet medical needs (e.g., wound healing, dry eye disease, alopecia, or skin damage or aging), and these options are described in this application. Summary of the Invention [Means for solving the problem]

[0004] Abstract The methods and compositions of the present disclosure advantageously utilize novel anucleated platelets or platelet-like cells or platelet variants (collectively referred to as "PLCs" (or singular "PLC")) or derivatives thereof or lysates thereof, or platelet-rich plasma derived therefrom, to fulfill the unmet need for the treatment, repair, or amelioration of diseases, disorders, or injuries related to osteoarthritis, tendon, ligament, bone repair, wound healing or wound healing-related disorders, dry eye, alopecia, or skin rejuvenation or regeneration, where adequate or consistent treatment is not available by conventional means (e.g., the use of platelets).

[0005] Advantageously, PLC or its derivatives, or PRP derived therefrom, are stable in quality and composition and can be produced relatively inexpensively compared to that obtained from platelets derived from human donors. More importantly, there is no need to extract PRP from the donor's blood, thereby eliminating the need to puncture the patient for blood collection. Furthermore, PLC is easily prepared because it is harvested from a bioreactor or fluidic device, and can be administered in a less invasive manner than other treatment options (e.g., without surgery or complex medical procedures). The PLC or its derivatives or lysates produced by the disclosed methods can be easily scaled up, continuously delivered (not hindered by batch-to-batch variability), and relatively free of contaminants. They can be used as a supplement to donor-derived PRP and administered locally at or near the site of injury to treat, repair, or alleviate tissue injury or tissue injury-related diseases (such as, but not limited to, bone-related disorders (e.g., osteoarthritis) or dry eye disease), to treat or alleviate regenerative diseases or disorders related thereto, or as a cosmetic to treat or alleviate skin aging or hair loss.

[0006] Thus, in some embodiments, compositions containing PLC or a derivative thereof or a lysate thereof, or PRP derived therefrom, or precursor cells that produce PLC or a derivative thereof or PRP derived therefrom, are provided that can be administered directly to or near the site of tissue injury to promote healing of injured tissue (such as, but not limited to, tendon, ligament, muscle, joint, or other musculoskeletal injuries). In some embodiments, compositions containing PLC or a derivative thereof or a PRP derived therefrom, or precursor cells that produce PLC or a derivative thereof or PRP derived therefrom, are administered locally or topically to the site of injury or near the site of damage (e.g., into the knee for the treatment of osteoarthritis), or under aging skin or near the site for skin or hair regeneration or rejuvenation. In some embodiments, the PLC or a derivative thereof, or precursor cells that produce PLC or a derivative thereof or PRP derived therefrom, are in the form of a lysate (i.e., the PLC or a derivative thereof, or precursor cells that produce PLC or a derivative thereof or PRP derived therefrom are concentrated, and the lysate is prepared from the concentrate). In some embodiments, PLC or lysate is concentrated and diluted with carrier, diluent, or buffer solution before use, or donor-derived PRP as disclosed herein.In some embodiments, the patient (i.e., subject) who is subjected to such treatment is a cancer patient who has been subjected to chemotherapy or other cancer treatment that leads to tissue injury or damage or hair loss, but the treatment of the present disclosure is not for cancer itself.In some embodiments, the treatment is for a patient who does not have cancer.

[0007] In some embodiments, PLC or a derivative thereof or a lysate thereof is enriched with growth factors or agents that stimulate the release of growth factors from PLC or a derivative thereof, or is mixed with platelet-rich plasma from a subject in a combination optimal for the subject.

[0008] PLC or its derivatives are produced using one or more tools and technologies, such as bioreactors or fluidic devices. Bioreactors or fluidic devices may include, but are not limited to, shear stress, mechanical strain, and pulsed electromagnetic field bioreactors, large-scale stirred tank bioreactors, automated bioreactors, rotating-wall bioreactors (RWBs), and rocking motions found in wave bioreactors, and organ chip bioreactors. Other bioreactor configurations capable of continuous perfusion operation, such as packed-bed bioreactors (PBBs), fluidized-bed bioreactors (FBBs), or PBBs or FBBs using microcarriers, CultiBag bioreactors, and membrane bioreactors (such as hollow fiber bioreactors (HFBs)), are also contemplated for producing the PLC / EVs or their derivatives of the present disclosure. Operation of the bioreactor may require connection to an internal or external cell retention device on a recycle line via centrifugation, sedimentation, ultrasonic separation, or microfiltration using spin filters, alternating tangential flow (ATF) filtration, or tangential flow filtration (TFF), or in vivo bioreactors, which are pockets within the body where biomaterials (e.g., PLC or its derivatives or the progenitor cells from which they are derived) are implanted at the site of need and incubated for extended periods. Within these pockets (e.g., bone tissue or muscle flaps), the implants harness the body's regenerative capabilities to recover from disease or injury. Non-limiting examples of bioreactors are described, for example, in the patent application Ser. No. 09 / 029,493, filed on Dec. 14, 2002, entitled "Simultaneous Welding of Cells and Cellular Implants." No. 62 / 981,373), and elsewhere (e.g., the tools and technologies (e.g., bioreactors or fluidic devices) disclosed in U.S. Patent Nos. 9,795,965; 10,343,163; 9,763,984; 9,993,503; and 10,426,799; U.S. Patent Application Publication No. 20180334652; PCT Application Nos. PCT / US2018 / 021354; PCT / US2019 / 012437; PCT / US19 / 040021; U.S. Patent Application No. 16 / 730,603, each of which is incorporated by reference in its entirety). Also, any known or unknown bioreactor or microfluidic device capable of routinely producing PLC or derivatives is contemplated for use in the present disclosure.

[0009] In some embodiments, the source of PLC or a derivative thereof, or a lysate thereof, or PRP derived therefrom, or the precursor cells from which PLC or a derivative thereof, or PRP derived therefrom, is autologous (i.e., they are produced, for example, using CD34+ progenitor cells from an individual in need of PLC-based treatment), and the progenitor cells are cultured in a bioreactor to produce PLC or a derivative thereof, which can be used as is, i.e., PLC or a derivative thereof, or a lysate thereof, or PRP derived therefrom (i.e., PRP enriched in PLC). In some embodiments, the source of PLC or a derivative thereof, or a lysate thereof, or PRP derived therefrom is iPSCs or iPSC-derived megakaryocytes.

[0010] Some other advantages offered by methods and compositions comprising PLC or its derivatives or lysates thereof, or PRP derived therefrom, are that they are allogeneic in nature, minimizing the risk of immune response; they are non-cancerous (i.e., they do not exhibit uncontrolled growth or tumor formation in vivo); and they are enriched in growth factors, such as, but not limited to, fibronectin, vitronectin, and sphingosine 1 phosphate (which facilitates the wound healing process). Additionally, PLC or its derivatives or lysates thereof, or PRP derived therefrom, or the precursor cells that make PLC or its derivatives or lysates thereof, or PRP derived therefrom, may reduce or eliminate adverse side effects induced, for example, by anti-inflammatory agents, opioids, or other drugs.

[0011] In some embodiments, the present disclosure provides a method of treating an injured subject, comprising administering to the subject a therapeutic amount of a composition comprising a PLC or derivative thereof or lysate thereof of the present disclosure, or PRP derived therefrom, thereby ameliorating, treating, or repairing the injury (e.g., osteoarthritis; tendon, ligament, bone repair, wound healing, or wound healing-related disorder), or assisting in the regeneration / rejuvenation of skin or hair (alopecia). In some embodiments, the method comprises administering a second or third therapeutic agent.

[0012] In some embodiments, the present disclosure provides a pharmaceutical composition comprising PLC, a derivative thereof, a lysate thereof, or PRP derived therefrom, or PLC, a derivative of PLC, or precursor cells that produce PLC, a derivative thereof, a lysate thereof, or PRP derived therefrom, and one or more pharmaceutically acceptable fillers, carriers, or excipients. In some embodiments, the pharmaceutical composition further comprises a second and a third therapeutic agent.

[0013] In some embodiments, the present disclosure provides non-natural extracellular vesicles (EVs) produced in vitro as a mixture with PLC. EVs include microvesicles (MVs) and exosomes with diameters of 200 to 1000 nm, which carry a wide variety of biologically active molecules (such as proteins, lipids, and RNA) on their surface or within their lumen. Each component in the mixture (i.e., PLCs, microvesicles, and exosomes) can be isolated from the mixture based on, for example, their size. Extracellular vesicles (EVs) have been implicated in stimulating wound healing, regenerating bone tissue to affect bone regeneration and repair processes, or regenerating skin or hair, indicating that EVs can confer wound healing, tissue regeneration, anti-apoptotic, or anti-inflammatory effects, for example, by transporting RNA and protein cargo. EVs also function as transport and delivery systems for bioactive molecules and play roles in hemostasis and thrombosis, inflammation, malignant infection propagation, angiogenesis, and immunity. Thus, in some embodiments, EVs may complement PLC or its derivatives, and their combined use further enhances the avenues for PLC-based therapeutic applications.

[0014] In some embodiments, the EVs of the present disclosure include exosomes, ranging in diameter from approximately 65 nm to about 10 μm, carrying a wide variety of molecules (such as proteins, lipids, and RNA) on their surface or within their lumen. Exosomes play a role in stimulating tissue regeneration in numerous in vitro and in vivo models, demonstrating that exosomes can confer proangiogenic, proliferative, anti-apoptotic, and anti-inflammatory effects through the transport of RNA and protein cargo. Thus, in some embodiments, exosomes further enhance the means for PLC-based therapeutic applications. In some embodiments, PLC or a derivative thereof or a lysate thereof, PRP derived therefrom, PLC or a derivative thereof, or precursor cells producing PLC or a derivative thereof or PRP derived therefrom, are administered in combination with extracellular vesicles (EVs), and the EVs are produced as a mixture with PLC but can be substantially isolated from the PLC due to their smaller size. Thus, in one embodiment, microvesicles or exosomes, alone or in combination with PLC or its derivatives, can be used for the stimulation of wound healing, the regeneration of bone tissue to affect bone regeneration and repair processes, or in the regeneration of skin or hair, for example, through the action of transporting RNA or transport agents (e.g., wound healing agents, tissue regeneration agents, anti-apoptotic agents, or anti-inflammatory agents, etc.) or protein cargo, thereby further enhancing the means of PLC-based therapeutic applications to impart pro-angiogenic, proliferative, anti-apoptotic, and anti-inflammatory effects that may be desirable in patients undergoing PLC-based treatments disclosed herein.

[0015] In some embodiments, megakaryocyte precursors, megakaryocytes, proplatelets, or preplatelets derived from induced pluripotent stem cells (iPSCs) that produce platelet-like cells (PLCs) and EVs (i.e., microvesicles or exosomes, or a combination thereof) can be genetically engineered to express a nucleic acid encoding a protein of interest (e.g., a wound-healing agent, a tissue-regenerating agent, an anti-apoptotic agent, or an anti-inflammatory agent) before passing through a bioreactor or fluidic device. In some embodiments, PLCs and / or EVs can be genetically engineered once such cells have passed through a bioreactor or fluidic device. Thus, in some embodiments, genetic modifications can be made 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 PLCs and / or EVs associated with PLC and / or EV production. Genetic engineering of megakaryocytes or megakaryocyte precursors differentiated from genetically engineered human pluripotent stem cell (hPSC) cells or cell lines, whereby megakaryocytes or megakaryocyte progenitor cells express a protein or polypeptide of interest, is also contemplated by the present disclosure. In some embodiments, PLCs and / or EVs, or derivatives thereof, differentiated from genetically engineered progenitor cells (e.g., megakaryocytes or megakaryocyte progenitor cells) deliver a protein of interest (e.g., a wound-healing agent, a tissue-regenerating agent, an anti-apoptotic agent, or an anti-inflammatory agent, etc.) systemically, or to a first diseased location (generally the diseased site where the PLCs and / or EVs (or genetically engineered versions thereof) were administered), or to a second diseased location different from the site where the PLCs and / or EVs, or derivatives thereof, were administered. Examples of such genetically engineered induced pluripotent stem cells or PSC-derived megakaryocytes that produce PLCs and / or EVs (i.e., genetically engineered PLCs / EVs or derivatives thereof) are disclosed in U.S. co-pending patent application Ser. Nos. 17 / 213,552 and 17 / 213,796, respectively, which are incorporated by reference in their entireties.Thus, in some embodiments, engineered PLCs (ePLCs) can be produced by genetically engineering PLC-producing progenitor cells such that the ePLCs express exogenous genes of interest, such as wound healing agents, tissue regeneration agents, anti-apoptotic agents, anti-inflammatory agents, anti-hormonal agents, or immunomodulatory agents, the enrichment of which would broadly complement PLC-based treatments.

[0016] It may be desirable to administer other compounds to the patient, such as, but not limited to, corticosteroids, tetrasubstituted pyrimidopyrimidines, 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, meclofenamate sodium, meloxicam, oxaprozin, sulindac, and tolmetin), COX-2 inhibitors (e.g., rofecoxib, celecoxib, valdecoxib, and lumiracoxib), glucocorticoid receptor modulators, or DMARDs. The combination therapies of the present disclosure are particularly useful for the treatment of immunoinflammatory disorders in combination with other agents (either biologics or small molecules) that modulate the immune response to positively impact the treatment of 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 enhance the action of anti-inflammatory cytokines (such as IL-10) and agents that inhibit the activity of pro-inflammatory cytokines (such as IL-6, IL-1, IL-2, IL-12, IL-15, or TNF-alpha). Agents that inhibit TNF-alpha include etanercept, adalimumab, infliximab, and CDP-870. In this example (an example of an agent that blocks the effects of TNF-alpha), combination therapy reduces cytokine production, while etanercept or infliximab acts on the remaining portion of the inflammatory cytokine, enhancing treatment. Small molecule immunomodulatory agents include, for example, p38 MAP kinase inhibitors (such as VX702, SCIO469, doramapimod, RO30201195, SCIO323), TACE inhibitors (such as DPC333), ICE inhibitors (such as pranalcasan), and IMPDH inhibitors (such as mycophenolate and merimepodib).Advantageously, when one or more of these compounds are administered together with PLC or a derivative thereof, the concentration of the co-administered compound, when included in the combined treatment, is expected to be reduced or decreased, such that any adverse known and unknown side effects that would occur if the compound were not administered with PLC are reduced by the co-administration of the compound.

[0017] In some embodiments, the present disclosure provides kits comprising the disclosed PLC or a derivative thereof or a lysate thereof or PRP derived therefrom, or precursor cells that produce PLC or a derivative thereof or a PRP derived therefrom or a derivative of PLC.

[0018] In some embodiments, the present disclosure provides a composition for treating a symptom in a subject, the composition comprising platelet-like cells (PLCs) or derivatives thereof and platelet-rich plasma (PRP) derived from a subject. In some embodiments, the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, an antihormonal agent, or an immunomodulatory agent, or a combination thereof. In some embodiments, the composition is diluted to a physiological concentration in a carrier, the carrier comprising a diluent or excipient. In some embodiments, the carrier is plasma, a plasma substitute, or Plasmalyte. In some embodiments, the PLCs or derivatives thereof are free of red blood cells, hemoglobin content, or white blood cells. In some embodiments, the composition further comprises extracellular vesicles (EVs). In some embodiments, the composition is formulated for application to an injury site for therapeutic use. In some embodiments, the composition further comprises another therapeutic agent. [Brief explanation of the drawings]

[0019] BRIEF DESCRIPTION OF THE DRAWINGS The present disclosure will now be described in detail in the following detailed description with reference to a number of drawings, in which like reference numerals refer to like parts throughout the several views, and in which:

[0020] [Figure 1-1] 1A-1B show the profile of growth and angiogenic factors in PLCs compared to donor platelets. [Figure 1-2] 1A-1B show the profile of growth and angiogenic factors in PLCs compared to donor platelets.

[0021] [Figure 2] FIG. 2 shows a schematic diagram of the design of PLC treatment of osteoarthritis in rats.

[0022] [Figure 3-1] Figures 3A-3F show that osteoarthritis-induced rats treated with PLC showed robust recovery from osteoarthritis approximately 8 weeks after treatment with PLC compared to untreated animals. Rats were administered a single dose on day 18. Figures 3A-3B show PLC-treated rats after 2 weeks of treatment. Figures 3C-3D show PLC-treated rats after 4 weeks of treatment. Figures 3E-3F show PLC-treated rats after 8 weeks of treatment. [Figure 3-2] Figures 3A-3F show that osteoarthritis-induced rats treated with PLC showed robust recovery from osteoarthritis approximately 8 weeks after treatment with PLC compared to untreated animals. Rats were administered a single dose on day 18. Figures 3A-3B show PLC-treated rats after 2 weeks of treatment. Figures 3C-3D show PLC-treated rats after 4 weeks of treatment. Figures 3E-3F show PLC-treated rats after 8 weeks of treatment. [Figure 3-3] Figures 3A-3F show that osteoarthritis-induced rats treated with PLC showed robust recovery from osteoarthritis approximately 8 weeks after treatment with PLC compared to untreated animals. Rats were administered a single dose on day 18. Figures 3A-3B show PLC-treated rats after 2 weeks of treatment. Figures 3C-3D show PLC-treated rats after 4 weeks of treatment. Figures 3E-3F show PLC-treated rats after 8 weeks of treatment. [Figure 3-4]Figures 3A-3F show that osteoarthritis-induced rats treated with PLC showed robust recovery from osteoarthritis approximately 8 weeks after treatment with PLC compared to untreated animals. Rats were administered a single dose on day 18. Figures 3A-3B show PLC-treated rats after 2 weeks of treatment. Figures 3C-3D show PLC-treated rats after 4 weeks of treatment. Figures 3E-3F show PLC-treated rats after 8 weeks of treatment. [Figure 3-5] Figures 3A-3F show that osteoarthritis-induced rats treated with PLC showed robust recovery from osteoarthritis approximately 8 weeks after treatment with PLC compared to untreated animals. Rats were administered a single dose on day 18. Figures 3A-3B show PLC-treated rats after 2 weeks of treatment. Figures 3C-3D show PLC-treated rats after 4 weeks of treatment. Figures 3E-3F show PLC-treated rats after 8 weeks of treatment. [Figure 3-6] Figures 3A-3F show that osteoarthritis-induced rats treated with PLC showed robust recovery from osteoarthritis approximately 8 weeks after treatment with PLC compared to untreated animals. Rats were administered a single dose on day 18. Figures 3A-3B show PLC-treated rats after 2 weeks of treatment. Figures 3C-3D show PLC-treated rats after 4 weeks of treatment. Figures 3E-3F show PLC-treated rats after 8 weeks of treatment. DETAILED DESCRIPTION OF THE INVENTION

[0023] While the above drawings illustrate embodiments of the disclosure herein, other embodiments are contemplated, as indicated in the discussion. This disclosure presents exemplary embodiments for purposes of illustration, not limitation. Numerous other modifications and embodiments may be devised by those skilled in the art that fall within the principles of the disclosed embodiments. Detailed Description

[0024] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide those of ordinary skill in the art with general definitions of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

[0025] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals exposed to the dosages and concentrations employed. Often, the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers (such as phosphate, citrate, and other organic acid buffers); antioxidants (including ascorbic acid); low molecular weight (less than about 10 residues) polypeptides; proteins (such as serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (such as polyvinylpyrrolidone); plasma or plasma substitutes, dextrans and hydroxyethyl starch, perfluorocarbons, and stroma-free hemoglobin; 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 non-ionic surfactants (such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®).

[0026] The term "derivative," as used herein, refers to genetically engineered PLCs (ePLCs) or genetically engineered extracellular vesicles (eEVs), or a combination thereof, for therapeutic use, and includes PLC precursor cells (e.g., pluripotent stem cells engineered in such a manner that the PLCs or extracellular vesicles produced by these PLC / EV precursor cells produce a molecule of interest in the PLC, the extracellular vesicles, or both, or any other modification described herein). Derivatives also include bioconjugates of PLCs and extracellular vesicles, or bioconjugates of engineered PLCs and extracellular vesicles. Derivatives also include cargo-carrying PLCs and extracellular vesicles, or cargo-carrying engineered PLCs and extracellular vesicles. For example, PLCs or extracellular vesicles can first be subjected to genetic engineering, and then their cargo-carrying ability is utilized. In other words, the term derivatives includes any modified, genetic, chemical, or combination thereof, substance of native PLCs, genetically engineered PLCs, extracellular vesicles, or genetically engineered extracellular vesicles.

[0027] As used herein, "progenitor cells" refers to IPSC-derived cells (preMK, MK, proplatelet, preplatelet, etc.). Progenitor cells also include "pluripotent stem cells," which include embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells, as well as other stem cells that have the potential to form cells from all three germ layers of the body, regardless of the method by which the pluripotent stem cells are derived. Pluripotent stem cells are functionally defined as stem cells that can have 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., the ability to differentiate into cell types of ectoderm, mesoderm, and endoderm); or (c) the ability to express one or more markers of embryonic stem cells (e.g., the expression of Oct4, 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 "megakaryocyte precursors" (preMKs), which refer to mononuclear hematopoietic cells of the megakaryocyte lineage, the precursors of which become mature megakaryocytes. Megakaryocyte precursors are typically found in, but are not limited to, bone marrow and hematopoietic locations, but can also be generated from pluripotent stem cells, such as by further differentiation of hemogenic endothelial cells derived from pluripotent stem cells.

[0028] The term "megakaryocyte precursor" (preMK), as used herein, refers to a mononuclear hematopoietic cell of the megakaryocyte lineage, the precursor to which becomes a mature megakaryocyte. Megakaryocyte precursors are typically found in, but are not limited to, bone marrow and hematopoietic locations, but can also be generated from pluripotent stem cells, such as by further differentiation of hemogenic endothelial cells derived from pluripotent stem cells.

[0029] The term "induced pluripotent stem cells" (iPS cells or iPSCs) refers to a type of pluripotent stem cell generated by reprogramming somatic cells through the expression of a combination of reprogramming factors. iPSCs can be generated 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.

[0030] "Agonist-activated" cell receptor or ligand activation is induced by receptor-specific agonists. Agonists activate cells by binding to their respective receptors or ligands on the cells.

[0031] As used herein, "variant" refers to a manifestation of structural diversity, deviation, or difference, and includes PLC or its derivatives or PLC, or microsomes, exosomes, vesicles, or any other product produced by culturing megakaryocytes in a bioreactor.

[0032] "PLC" or artificial platelets, as used interchangeably herein, refer to novel, non-naturally occurring, anucleated platelets or platelet-like cells that differ in structure from naturally occurring, bone marrow-derived platelets (i.e., their native counterparts). PLC also includes platelet variants as defined elsewhere.

[0033] As used herein, "variant" or "variants," as used interchangeably herein, refers to the manifestation of structural diversity, deviations, or differences between PLC and donor platelets. As a non-limiting example, a variant may have an average of less than 2% of the CD63 receptor (i.e., (CD63 <平均2% )) compared to reference resting bone marrow-derived platelet cells with an average of more than 2% CD63 receptors (i.e., CD63 >平均2% In some embodiments, the variants contain an average of more than 80% of the CD36 receptor (i.e., (CD36 >平均80% )) compared to reference resting bone marrow-derived platelet cells with an average of less than 10% CD36 receptors (i.e., CD36 <平均80% Alternatively, the variant contains an average of more than 95% of the CD42b receptor (i.e., (CD42b >平均95% )) compared to reference resting bone marrow-derived platelet cells that have an average of less than 95% CD42b receptors (i.e., CD42b <平均95% Alternatively, the variant contains an average of more than 90% of the GPVI receptors (i.e., (GPVI >平均90% On average, less than 90% of the glycoprotein VI receptors (i.e., (GPVI)) were expressed in the 100% ... <平均90% )). The term variant also includes PLC structural configurations comparable to those of naturally occurring bone marrow-derived platelets, either resting or activated. For example, PLC and donor platelets may have m% CD36, or n% CD42a receptor, or o% CD42a-bd receptor, or p% CD61 receptor, or q% CD62p receptor, or x% CD63 receptor, where m%, n%, o%, p%, q%, and x% are identical (i.e., equal in value) between the PLC and bone marrow-derived platelets. In other words, the structure may be identical to that of the donor platelets, but may further exhibit the advantages of the PLC variants disclosed herein in the present disclosure.

[0034] "Comprises," "comprising," "containing," and "having," etc., can have the meaning ascribed to them in U.S. patent law and can mean "includes," "including," etc. (e.g., a composition "comprising" X can consist exclusively of X or can include some addition (e.g., X+Y)); "consisting essentially of" or "consists essentially of," etc., have the meaning ascribed to them in U.S. patent law, and the terms are open-ended, allowing for the presence of more than the recited terms so long as the basic or novel characteristics of the recited terms are not altered by the presence of more than the recited terms, but precludes prior art embodiments.

[0035] Unless otherwise stated or clear from context, as used herein, the term "or" is understood to be inclusive. Unless otherwise stated or clear from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural.

[0036] Unless otherwise specified or clear from the context, when used herein, the term "about" is understood to be within the normal tolerance in the art (e.g., within 2 standard deviations of the mean). "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0037] As used herein, "mean" refers to a number that indicates the central or typical value in a data set, particularly the mode, median, or (most commonly) average calculated by dividing the sum of the values ​​in the data set by the number of values ​​in the data set. Mean also refers to a single value (such as the mean, mode, or median) that summarizes or indicates the general significance of a unequal set of values.

[0038] As used herein, "non-natural" refers to something that is produced, created, or constructed by man, something that is artificial, or something that mimics something natural.

[0039] Routes of administration for various embodiments include, but are not limited to, local, regional, transdermal, nasal, and systemic administration (such as intravenous, intraarterial, intramuscular, subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal, intraarticular, ocular, otic, or oral administration) at the site requiring treatment (e.g., the knee for the treatment of osteoarthritis). As used herein, "systemic administration" refers to all non-cutaneous routes of administration and specifically excludes topical and transdermal routes of administration. Additional methods of administration of the disclosed PLC or derivatives thereof include intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intersternal injection and infusion.

[0040] The term "antagonist" as used herein refers to a substance that initiates a physiological response when combined with a receptor. An agonist activates cells by binding to its respective receptor or ligand on the cell. It can be any drug or entity that can inhibit the expression and activity of a protein, its polypeptide portion, or a polynucleotide. Thus, an antagonist can be engineered to prevent transcription, translation, post-transcriptional or post-translational processing, or otherwise inhibit the activity of a protein, polypeptide, or polynucleotide in any way, either by direct or indirect action. An antagonist can be, for example, a nucleic acid, a peptide, or any other suitable compound or chemical molecule, or any combination thereof. Furthermore, in indirectly attenuating the activity of a protein, polypeptide, or polynucleotide, it is understood that an antagonist can affect the activity of a cellular molecule, and a cellular molecule can act as a regulator of the protein, polypeptide, or polynucleotide itself. Similarly, an antagonist can affect the activity of a molecule, which itself is subject to control or regulation by the protein, polypeptide, or polynucleotide. The term "agonist activation," as used herein, refers to activation of a cellular receptor or ligand induced by a receptor-specific agonist.

[0041] "Donor platelets" refer to bone marrow-derived platelets that are physiologically produced in a mammal (e.g., a human). Donor PRP refers to PRP prepared from the blood of a donor.

[0042] As used herein, the term "subject" refers to a mammal, such as, but not limited to, a human, pig, horse, dog, or cat, or any other animal capable of suffering from a viral infection. Subjects include healthy populations, populations susceptible to infection with a pathogen, or patients suffering from a viral infection.

[0043] As used herein, the terms "drug," "pharmaceutical agent," or "compound" refer to a biological product or chemical entity, or a combination of biological products and chemical entities, administered in therapeutic amounts to a human to treat, prevent, or regulate a disease or condition. Biological products or chemical entities include, but are not limited to, antibodies or fragments thereof, low molecular weight compounds, and can also be larger compounds, such as oligomers of nucleic acids, amino acids, or carbohydrates (including, but not limited to, proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof).

[0044] The terms "drug," "therapeutic composition," or "therapeutic agent" can be used interchangeably and refer to a therapeutic agent. The drug can be selected from one or more of the following: proteins; peptides; aptamers; antibodies; or fragments thereof; chemicals; small molecules; nucleic acid sequences; and nucleic acid analogs. The nucleic acid sequence can be RNA or DNA, single-stranded or double-stranded, and can be selected from nucleic acids encoding proteins of interest, oligonucleotides, and nucleic acid analogs (e.g., peptide-nucleic acid (PNA), pseudocomplementary PNA (pc-PNA), locked nucleic acid (LNA), etc.). Such nucleic acid sequences 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 (e.g., RNAi, shRNAi, siRNA, micro-RNAi (mRNAi), antisense oligonucleotides, etc.). The protein and / or peptide or fragment thereof can be any protein of interest (e.g., but not limited to, a mutant protein; a therapeutic protein; a truncated protein), where the aforementioned proteins are normally absent or expressed at low levels in cells. The protein 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 agent can be applied to a medium that contacts the cell and induces its effect. Alternatively, the agent can be an intracellular agent, where a nucleic acid sequence is introduced into the cell and transcribed to produce the nucleic acid and / or protein upon environmental stimuli within the cell. In some embodiments, the agent is any chemical, entity, or moiety, including, but not limited to, synthetic and naturally occurring non-proteinaceous entities. In some embodiments, the agent is a small molecule having a chemical moiety. For example, the chemical moiety includes an unsubstituted or substituted alkyl, aromatic, or heterocyclyl moiety, including macrolides, leptomycin, and related natural products or analogs thereof.Agents can be known to have desired activities and / or properties, or can be selected from a diverse library of compounds.

[0045] The term "antibody," as used herein, refers to an immunoglobulin molecule that specifically binds to an antigen. The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody.

[0046] The terms "culture conditions" or "culture medium" or "medium" can be used interchangeably and refer to a medium for cell culture that contains nutrients that maintain cell viability and support cell expansion and maintenance or cell differentiation. In addition to the embodiments disclosed herein, cell culture media can contain any of the following appropriate combinations: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum substitutes, and other components (such as peptide growth factors). Appropriate cell culture media for cell types are known to those of skill in the art.

[0047] As used herein, the terms "treat," "treating," and "treatment" refer to the reduction or amelioration of a disorder and / or its associated symptoms. It is recognized that treatment of a disorder or condition does not require, although not preclude, the disappearance of the disorder, condition, or its associated symptoms.

[0048] As used herein, the term "PLC-enriched plasma" refers to enriched PRP provided by combining intact PLC or its derivatives, or precursor cells that produce PLC or derivatives, and lysates extracted therefrom, to enrich other plasma sources (such as donor-derived PRP). Optionally, this may contain growth factors, cytokines, or other agents from other sources that complement the therapeutic application of PLC or its derivatives.

[0049] As used herein, the term "or," where specified or clear from context, is understood to be inclusive. Unless specified otherwise or clear from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural.

[0050] The phrase "substantially" does not exclude "completely" (e.g., a composition that is "substantially free" of Y may be completely free of Y). If necessary, the phrase "substantially" may be omitted from the definitions of the present disclosure.

[0051] The term "extracellular vesicles (EVs)" as used herein collectively refers to microvesicles and exosomes, which are generally very small phospholipid vesicles (generally approximately 1 micron or less in diameter; microvesicles generally about 200-1500 nm or less in diameter; exosomes generally 20-200 nm or less in diameter) shed from megakaryocytes or other cells. Extracellular vesicles (EVs) may contain or transport materials, such as, but not limited to, nucleic acids (e.g., siRNAs), growth factors, proteins, or exogenous genetic material (e.g., for gene therapy), and may express extracellular markers of their parent cells. Megakaryocyte-derived EVs may play a role in multiple pathways, including hemostasis and inflammation, and in the treatment of various disorders, such as, but not limited to, malignant diseases (e.g., neoplasia), Alzheimer's disease, and tumor progression and development.

[0052] The term "cryopreservation medium" refers to a liquid medium (solution or suspension) that can protect the structure and metabolism of isolated cells from damage associated with a freezing event, either internal or external to the cells, and that is safe for infusion or injection into humans. This term also refers to a medium (solution or suspension) containing components, including cryopreservatives, that have been determined or are known to be safe for infusion or injection into humans. Preferably, the medium (solution or suspension) and the agent, component or element of the medium are approved by U.S. regulatory authorities for infusion or injection into humans (e.g., histidine (25-50 mM)). A "cryoprotectant" is an agent that can confer some degree of cryoprotection to the structure and metabolism of cells upon freezing. Cryoprotectants within the scope of the present disclosure include arabinogalactan and its biological and functional equivalents, glycerol, propylene glycol, and albumin (e.g., human serum albumin), plasma, or serum.

[0053] The term "treatment" is intended to encompass any form of treatment, prophylaxis, or diagnosis, and includes treatment for both the cure and prevention of disease. Thus, treatment of healthy patients shall be considered treatment. Treatment also covers the alleviation of symptoms in addition to the curative treatment of disease.

[0054] The compositions and methods of the present disclosure take advantage of the properties of PLC or its derivatives, including engineered PLC, such as, but not limited to, providing growth factors, cytokines, and other agents to offer a unique opportunity to maximize therapeutic outcomes and also minimize side effects in the treatment, repair, amelioration of diseases, disorders, or injuries related to osteoarthritis, tendon, ligament, bone repair, wound healing or wound healing-related disorders, dry eye, alopecia, or skin rejuvenation or regeneration, where adequate or consistent treatment is unavailable by conventional means.

[0055] Prior art protocols for the use of PRP enrichment are essentially multi-step processes, increasing the risk of contamination and resulting in inconsistent and unreliable results. For example, a typical PRP preparation involves the following steps: (1) collecting a small amount of venous blood (15–50 mL) from a patient's arm into a tube containing an anticoagulant; (2) the recommended temperature during processing is 21–24°C to prevent platelet activation; (3) centrifuging the blood at 1,200 rpm for 12 minutes; (4) separating the blood into three layers: an upper layer containing platelets and white blood cells, a thin white blood cell-rich middle layer (buffy coat), and a lower layer containing red blood cells; and (5) transferring the upper and middle buffy coat layers to empty, sterile tubes. The plasma is centrifuged again at 3,300 rpm for 7 minutes to aid in the formation of a soft pellet (erythrocytes and platelets) at the bottom of the tube; (6) the top two-thirds of the plasma is platelet-poor plasma and is discarded; (7) the pellet is homogenized in the bottom one-third (5 mL) of the plasma to create injectable PRP.

[0056] The present disclosure eliminates many of the prior art steps involved in PRP preparation, thereby minimizing the risk of contamination or impurities. Most importantly, it does not require the collection of venous blood, which is often a source of contamination (e.g., viruses such as HIV) unless rigorously analyzed. In other words, the present disclosure, at a minimum, does not require blood collection or separation into different components before platelets can be isolated. This is achieved by culturing the progenitor stem cell population to conditions such that they substantially differentiate into mature megakaryocytes. The mature megakaryocytes are then cultured in a bioreactor or fluidic device, where a gradient in the bioreactor mimics the endogenous platelet-producing environment to generate PLCs or their derivatives. PLCs or their derivatives are then harvested from the bioreactor in sufficient amounts to be used. In some embodiments, PLCs or their derivatives or their lysates can be used by themselves or in combination with EVs. In some embodiments, PLCs or their derivatives or their lysates can be used to supplement donor platelets in platelet-rich plasma (i.e., PLCs or genetically engineered PLCs are mixed with donor-based PRP). In some embodiments, PLC or a derivative thereof can be used in combination with an EVS or other drug disclosed herein.

[0057] PLC or its derivatives or lysates thereof or PRP derived therefrom, or the precursor cells that make PLC or its derivatives or PRP derived therefrom, are rich in growth factors and cytokines and can be used in intact, liquid, paste form, or mixed with other agents (such as, but not limited to, gels, ointments, creams, or other emulsifiers, acceptable diluents, carriers, or excipients).

[0058] In some embodiments, the present disclosure further provides a pharmaceutical composition comprising the PLC of the present invention, a derivative thereof, a lysate thereof, or PRP derived therefrom, or PLC or a PLC derivative, or precursor cells that produce PLC, a derivative thereof, a lysate thereof, or PRP derived therefrom. In some embodiments, the pharmaceutical composition comprises (1) PLC, a derivative thereof, a lysate thereof, or PRP derived therefrom, or PLC or a PLC derivative, or precursor cells that produce PLC, a derivative thereof, or PRP derived therefrom, and (2) a pharmaceutically acceptable filler, carrier, or excipient. In some embodiments, the pharmaceutical composition comprises (1) PLC, a derivative thereof, a lysate thereof, or PRP derived therefrom, or PLC or a PLC derivative, or precursor cells that produce PLC, a derivative thereof, or PRP derived therefrom, (2) a pharmaceutically acceptable filler, carrier, or excipient, and, optionally, (3) at least one additional therapeutic agent. In some embodiments, the pharmaceutical composition comprises (1) PLC or a derivative thereof or a lysate thereof or PRP derived therefrom or PLC or a PLC derivative, or precursor cells that produce PLC or a derivative thereof or PRP derived therefrom, (2) a pharmaceutically acceptable bulking agent, carrier, or excipient, and, optionally, (3) at least one additional therapeutic agent and enrichment agent.

[0059] A composition containing PLC or a derivative thereof or a lysate thereof, or PRP derived therefrom, is used in combination with a polysaccharide (e.g., natural polysaccharides (e.g., hyaluronic acid, hydroxypropyl cellulose, karaya gum (KG), guar gum (GUG), or gellan gum (GEG))), a dermal filler (e.g., Juvederm®, Juvederm® Ultra Plus, Perlane®, Belotero®, Restylane®, etc.), semi-synthetic or synthetic polysaccharides, and synthetic polymers (e.g., poly(7-oxanorbornene-2-carboxylate), F127, or poly(lactic-co-glycolic acid) (PLGA)), sodium citrate, calcium chloride, proteoglycans, adenine, guanine, cytosine, thiamine, progenitor stem cells or derivatives thereof, vitamins, retinol, retinoic acid, retinol palmitate, acetate (e.g., tocopheryl acetate), acetate), phosphates (e.g., sodium ascorbyl phosphate), D-panthenol, peptides, recombinant growth factors, micronized human-identical hormones, amino acids, botanical extracts, antioxidants, lipoic acid, DMAE, collagen, GAGs, trace elements, minerals, proteases, ceramides, polysaccharides, algae, marine extracts, monocytes, or combinations thereof.

[0060] Additional therapeutic agents include, but are not limited to, those described elsewhere in this disclosure. A composition comprising PLC or a derivative thereof or a lysate thereof, or PRP derived therefrom, or PLC or a PLC derivative, or precursor cells that produce PLC or a derivative thereof, or PRP derived therefrom, can be administered locally (e.g., to an osteoarthritic knee), parenterally (i.e., by injection, subcutaneously, intramuscularly, intravenously, intradermal, intrathecal, and epidurally), or topically, ophthalmically, or by inhalation, or rectally, or vaginally, or sublingually.

[0061] Therapeutic compositions and formulations thereof containing PLC or a derivative thereof or a lysate thereof, or PRP or a PLC derivative derived therefrom, or precursor cells that produce PLC or a derivative thereof or PRP used in the present disclosure, are prepared in the form of a lyophilized formulation or aqueous solution for storage by mixing PLC or a derivative thereof or a lysate thereof, or PRP derived therefrom, or PLC or a PLC derivative, or precursor cells that produce PLC or a derivative thereof or PRP derived therefrom, of the desired purity, with a pharmaceutically acceptable carrier, excipient, or stabilizer as needed (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers (such as acetate buffer, Tris buffer, phosphate buffer, citrate buffer, and other organic acid buffers); antioxidants (including ascorbic acid and methionine); preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens (such as methylparaben or propylparaben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins (such as serum albumin, gelatin, or immunoglobulins, etc.); 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); tonicity agents (such as trehalose and sodium chloride); sugars (such as sucrose, mannitol, trehalose, or sorbitol); surfactants (such as polysorbates); salt-forming counterions (such as sodium); metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants (such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG)).Other pharmaceutically acceptable carriers may be, but are not limited to, binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, calcium hydrogen phosphate, etc.), lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, 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, alcohol, polyethylene glycol, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone. Pharmaceutical formulations to be used for in vivo administration are generally sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0062] The formulations herein may also contain more than one active compound, preferably compounds whose activities complement each other without adversely affecting each other, as needed for the particular indication being treated. For example, in addition to PLC or its derivatives or lysates thereof, PRP derived therefrom, or PLC or its derivatives, or precursor cells that produce PLC or its derivatives or PRP derived therefrom, it may be desirable to include additional injury-healing agents (e.g., anti-inflammatory agents or opioid drugs) in a single formulation. Alternatively or in addition, the composition may further contain cytotoxic agents, cytokines, growth inhibitors, antihormonal agents, and / or cardioprotective agents. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0063] The active ingredient (i.e., PLC or its derivatives or lysates thereof, or PRP derived therefrom) can also be entrapped in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, respectively) prepared by coacervation techniques or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0064] The active ingredient (i.e., PLC or its derivatives or lysates, or PRP derived therefrom) can also be delivered into a bioactive scaffold made from natural (e.g., protein-based) or synthetic (polymer- or ceramic-based) biomaterials. In addition to PLC or its derivatives or lysates, or PRP derived therefrom, the bioactive scaffold can optionally contain growth factors and other bioactive molecules. Some examples of these include epidermal growth factor, TGF-alpha, TGF-beta, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, insulin-like growth factor, keratinocyte growth factor, and bone morphogenetic protein. Growth factors can also be introduced into the scaffold before or after the introduction of PLC or its derivatives or lysates, or PRP derived therefrom.

[0065] Sustained-release preparations can be prepared.Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing PLC or its derivatives or its lysate or PRP derived from them or PLC or PLC derivatives, or precursor cells that produce PLC or its derivatives or PRP derived from them, and the aforementioned matrices are in the form of shaped articles (for example, films or microcapsules).Examples of sustained-release matrices include polyester, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate)), and poly-D-(-)-3-hydroxybutyric acid. Biocompatible materials that may be present in the hydrogel include, for example, permeable compositions or forms (such as polyvinyl alcohol, polyvinylpyrrolidone and polyacrylamide, polyethylene oxide, poly(2-hydroxyethyl methacrylate)); natural polymers (such as polysaccharides, gums, and starches); poly[alpha(4-aminobutyl)]-1-glycolic acid, polyethylene oxide, polyorthoesters, silk-elastin-like polymers, alginates, EVAc (poly(ethylene-co-vinyl acetal)), Examples of suitable biocompatible materials include cellulose, cellulose acetate, cellulose acetate copolymers ...In one embodiment, the hydrogel comprises poloxamer, polyacrylamide, poly(2-hydroxyethyl methacrylate), carboxyvinyl polymer (e.g., Carbopol 934, Goodrich Chemical Co.), cellulose derivatives (e.g., methyl cellulose, cellulose acetate, and hydroxypropyl cellulose), polyvinylpyrrolidone, or polyvinyl alcohol, or a combination thereof. In some embodiments, the hydrogel comprises collagen (eg, hydroxylated collagen), fibrin, polylactic-polyglycolic acid, or a polyanhydride. Other examples include, but are not limited to, any biocompatible polymer, 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 oxidey)-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 (alginate, agarose, starch, fibrin, collagen, gelatin, chitin, glycosaminoglycans (e.g., hyaluronic acid, dermatan sulfate, and chondroitin sulfate), and the like. sulfate), and microbial polyesters (such as, for example, hydroxyalkanoates, such as copolymers of hydroxyvalerate and hydroxybutyrate)) and synthetic polymers (such as poly(orthoesters) and polyanhydrides, including homopolymers and copolymers of glycolide and lactide (e.g., poly(L-lactide, poly(L-lactide-co-D,L-lactide), poly(L-lactide-co-glycolide), polyglycolide, and poly(D,L-lactide), poly(pol)(D,L-lactide-coglycolide), poly(collidine lactate), and polycaprolactone)). While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for over 100 days, certain hydrogels release proteins for shorter time periods.If encapsulated PLC or its derivatives or lysates thereof, or PRP or PLC or PLC derivatives derived therefrom, or precursor cells that produce PLC or its derivatives or PRP derived therefrom, remain in the body for extended periods of time, they may denature or aggregate as a result of exposure to moisture at 37°C, which may result in loss of biological activity and altered activation.

[0066] PLC or its derivatives or their lysates, or PRP derived therefrom, or PLC or PLC derivatives, or precursor cells producing PLC or its derivatives or PRP derived therefrom, can be formulated in any form suitable for delivery to target cells / tissues. For example, PLC or its derivatives or their lysates, or PRP derived therefrom, or PLC or PLC derivatives, or precursor cells producing PLC or its derivatives or PRP derived therefrom, can be formulated as immunoliposomes. "Liposomes" are vesicles composed of various types of lipids, phospholipids, and / or surfactants that are useful for delivering drugs to mammals. The components of liposomes are generally arranged to form a bilayer similar to the lipid arrangement of biological membranes. Liposomes containing PLC or its derivatives or their lysates, or PRP derived therefrom, or PLC or PLC derivatives are described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al. al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO 97 / 38731, published October 23, 1997. Liposomes with improved circulation time are disclosed in U.S. Pat. No. 5,013,556.

[0067] Useful liposomes can be produced by reverse phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters with defined pore sizes to obtain liposomes with the desired diameter. Additional therapeutic agents are optionally contained within the liposomes. See Gabizon et al., J. National Cancer Inst. 81(19):1484(1989).

[0068] Formulations to be used for in vivo administration should be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0069] For the treatment of injuries (e.g., OA, regeneration of bone tissue to affect the process of bone regeneration and repair) or regeneration of aging skin or hair, in one embodiment, PLC or a derivative thereof or a lysate thereof, or PRP derived therefrom, or PLC or a PLC derivative, or precursor cells that produce PLC or a derivative thereof or PRP derived therefrom, are administered via any of the routes disclosed herein. The dosage administered via any of these routes is about 0.1 micrograms / m at a time. 2 ~approximately 10,000 micrograms / m 2 and generally administered once daily or once weekly for a total of 1, 2, 3, or 4 times, or multiple times as needed. Alternatively, the dosage range is about 0.1 micrograms / m 2 ~approximately 1000 micrograms / m 2 , approximately 0.1 micrograms / m 2 ~approximately 800 micrograms / m 2 , approximately 0.1 micrograms / m 2 ~approximately 600 micrograms / m 2 , approximately 0.1 micrograms / m 2 ~approximately 400 micrograms / m 2 , approximately 0.1 micrograms / m 2 ~approximately 500 micrograms / m 2, approximately 0.1 micrograms / m 2 ~approximately 300 micrograms / m 2 , approximately 0.1 micrograms / m 2 ~approximately 200 micrograms / m 2 , and approximately 0.1 micrograms / m 2 ~approximately 200 micrograms / m 2 To relieve or alleviate symptoms of injury or skin aging or a disease associated with injury or skin aging, the aforementioned doses can be administered 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 daily, weekly, or monthly (e.g., administered once or more than once daily, weekly, biweekly, three weeks, or monthly). Administration can continue at any of the disclosed intervals until remission of symptoms associated with injury or skin aging or a disease. Administration can continue after symptoms have remitted or been relieved, and such remission or relief is prolonged by continued administration.

[0070] The emulsifier may be natural or synthetic, including, but not limited to, cationic (e.g., benzalkonium chloride, benzethonium chloride), anionic (e.g., alkaline soaps (sodium or potassium oleate); amine soaps (triethanolamine stearate); surfactants (sodium lauryl sulfate, dioctyl sodium sulfosuccinate, sodium docusate)), and nonionic (e.g., sorbitan esters (Spans®), polyoxyethylene derivatives of sorbitan esters (Tweens®), or glyceryl esters).

[0071] Other agents that can be used as emulsifiers may include deoxycholic acid, diacetyl tartaric acid ester, egg yolk, glycerol, gum, Irish moss (carrageenan), lecithin, mono- and diglycerides, monosodium phosphate, monostearate, ox bile extract, propylene glycol, soap, taurocholic acid (or its sodium salt).

[0072] In some embodiments of the present disclosure, PLC or its derivatives or their lysates, or PRP derived therefrom, or precursor cells that produce PLC or its derivatives or PRP derived therefrom, are present in Plasmalyte. Plasmalyte is a family of balanced crystalloid solutions. Plasmalyte closely resembles human plasma in electrolyte content, osmolality, and pH. These solutions also have additional buffering capacity and contain anions such as acetate, gluconate, and lactate, which are converted to bicarbonate, CO2, and water.

[0073] In some embodiments, MKs and platelets are also derived from human induced pluripotent stem cells (iPSCs), hematopoietic stem cells (CD34 + Umbilical cord blood stem cells (UCB cells) (e.g., human CD34 + umbilical cord blood stem cells) (such as those disclosed in PCT applications based on co-pending U.S. Patent Application No. 16 / 975,918, filed June 25, 2020, and U.S. Provisional Patent Application No. 63 / 025,209, filed May 15, 2020, which are incorporated by reference in their entireties), CD34 + Mobilized peripheral blood cells (MPB cells) (e.g., CD34 + human mobilized peripheral blood), or CD34 + They can be derived from bone marrow cells. UCB cells are blood-derived multipotent stem cells that remain in the placenta and attached umbilical cord after birth. MPB cells are multipotent stem cells derived from volunteers whose stem cells are mobilized into the bloodstream by administering G-CSF or similar agents.

[0074] In some embodiments, the MKs and platelets may be derived from other stem cell types, including, but not limited to, mesenchymal stem cells (MSCs) (such as adipose-derived mesenchymal stem cells (AdMSCs)) or mesenchymal stem cells from other sources.

[0075] AdMSCs are derived from white adipose tissue, which is derived from mesoderm during embryonic development and is present in all mammalian species and distributed throughout the body. Due to their wide availability and ability to differentiate into other tissue types of mesoderm, including bone, cartilage, and muscle, adipose-ASCs may be useful in a wide variety of applications.

[0076] In the present disclosure, stem cell cultures can be maintained independent of embryonic fibroblast feeder cells and / or animal serum. In some embodiments, serum-free, feeder-free alternatives can be utilized in the methods of the present invention. Extracellular vesicles (EVs)

[0077] In some embodiments, the present disclosure includes microvesicles and exosomes (collectively referred to as extracellular vesicles (EVs)) or derivatives thereof from which a mixture of PLCs is produced. 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 can deliver important payloads to recipient cells (e.g., osteoarthritic knees or skin, or damaged organs or tissues, or for use in regenerative medicine), thereby further enhancing the means to supplement PLCs with additional growth factors or other molecules for, for example, the treatment or amelioration 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.

[0078] The disclosed EVs or their derivatives can be isolated and purified from a mixture containing the disclosed PLC, essentially separating them. Isolated or purified extracellular vesicles (EVs) or their derivatives can travel widely throughout the body, and when administered to a patient, they can exert significant therapeutic effects. EVs have fundamental immunomodulatory properties for the treatment or inhibition of diseases or disorders related to dry eye syndrome, osteoarthritis, tendon, ligament, and bone repair, wound healing or wound healing-related disorders, alopecia, or other injuries, or for skin rejuvenation or regeneration. EVs can also be used as drug delivery systems; they can cross biological barriers, including the blood-brain barrier and synovial membrane.

[0079] Advantageously, EVs or their derivatives can be internalized by recipient cells after receptor-ligand interactions, and a rich combination of bioactive molecules (such as proteins, bioactive lipids, and nucleic acids) from the cell of origin can be transferred along with proteins expressed on the EV surface.

[0080] In some embodiments, EVs or their derivatives can directly activate recipient cells (e.g., donor platelets) by acting as signaling complexes. For example, EVs or their derivatives can bind to platelets via P-selectin glycoprotein ligand-1 expressed on their surface, and EVs or their derivatives derived from neutrophils expressing Mac-1 can induce donor platelet activation in patients in need thereof. Such activation is advantageous because it facilitates the activation of exogenous mechanisms that can enhance the treatment or amelioration of diseases, disorders, or injuries related to dry eye, osteoarthritis, tendon, ligament, and bone repair, wound healing or wound healing-related disorders, alopecia, or skin rejuvenation or regeneration.

[0081] Compositions and methods comprising the disclosed extracellular vesicles (EVs) or derivatives thereof can be used in several therapeutic or cooperative therapies, such as the treatment or amelioration of diseases, disorders, or injuries associated with dry eye syndrome, osteoarthritis, tendon, ligament, or bone repair, wound healing or wound healing-related disorders, alopecia, or skin rejuvenation or regeneration, for example, for the delivery of genes, proteins, peptides, or nucleic acids for cell or gene therapy using vectors (e.g., adenovirus, lentivirus), to produce novel microvesicles or exosomes to deliver genes (e.g., for gene therapy), peptides (for growth factors), or nucleic acids (e.g., siRNA or microRNA). Packaging within extracellular vesicles (EVs) offers several advantages, such as protecting molecules from harmful cellular events that could neutralize naked genes. Engineered extracellular vesicles (EVs) can be used to deliver drugs to specific sites in damaged tissues, including, but not limited to, dry eye, osteoarthritis, tendons, ligaments, bone repair, wound healing or wound healing-related disorders, alopecia, or skin rejuvenation or regeneration.

[0082] In some embodiments, the isolated extracellular vesicle (EV) derivatives can then be stored until use by freezing at ultra-low temperatures (e.g., -80°C) in the presence of cryopreservatives such as dimethyl sulfoxide (DMSO) and glycerol used at optimal concentrations.

[0083] In some embodiments, the average diameter of extracellular vesicles (EVs) derived from the iPSC-derived platelet population is less than 50% of the diameter of extracellular vesicles (EVs) derived from a donor-derived platelet population having approximately the same number of platelets as the iPSC-derived platelet population. In some embodiments, the megakaryocytes or platelets have been genetically modified to contain a nucleic acid molecule encoding a therapeutic agent.

[0084] Extracellular vesicles (EVs) are subcellular particles consisting of a membrane lipid bilayer and intracellular contents. EVs isolated or purified from mixtures containing PLCs exert both anti- and pro-inflammatory functions and may have potential as drug delivery vehicles.

[0085] In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 4 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 3 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 2.5 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 1.5 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 1.0 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 0.9 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 0.8 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 0.7 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 0.6 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 0.5 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 0.4 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 0.3 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.1 μm and 0.2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.2 μm and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.3 μm and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.4 μm and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.5 μm and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.6 μm and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.7 μm and 1 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 0.8 μm and 1 μm.In some embodiments, the diameters of the extracellular vesicles (EVs) of the present invention are 0.9 μm and 1 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) of the present invention are 0.2 μm and 2 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) of the present invention are 0.3 μm and 2 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) of the present invention are 0.4 μm and 2 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) of the present invention are 0.5 μm and 2 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) of the present invention are 0.6 μm and 2 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) of the present invention are 0.7 μm and 2 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) of the present invention are 0.8 μm and 2 μm. In some embodiments, the diameters of the extracellular vesicles (EVs) of the present invention are 0.9 μm and 2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 1.0 μm and 2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 1.5 μm and 2 μm. In some embodiments, the diameter of the extracellular vesicles (EVs) of the present invention is 2.0 μm and 2.5 μm.

[0086] The extracellular vesicle (EV) derivatives can be conjugated to one or more cytotoxic agents by the mechanisms disclosed above. One or more cytotoxic agents can be absorbed into the extracellular vesicle (EV) derivatives by the mechanisms also disclosed above. Cytotoxic agents are also disclosed above. Diseases and disorders that can be cured or alleviated by the use of the EV derivatives alone or in combination with the disclosed PLC or its derivatives are also disclosed below.

[0087] In some embodiments, EVs can be developed for therapeutic use independently of PLC or its derivatives, regardless of modification (e.g., bioengineered or conjugated). For example, patients requiring treatment primarily involving microvesicles or their derivatives can be administered microvesicle-based treatment, exosome-based treatment, or a combination of both. For example, MVs or exosomes incorporating exogenous growth factors, cytokines, or siRNAs can be used to efficiently silence target MAPK genes in monocytes and lymphocytes, or to deliver growth factor siRNAs (e.g., VEGF-siRNAs) that target bone tissue regeneration and alopecia, for example, to affect the process of bone regeneration and repair. Advantageously, MVs can be used as a more efficient delivery vehicle for specific targeting of novel therapeutic agents without causing immunogenicity and adverse effects.

[0088] In some embodiments, EV-based treatment can be administered before PLC-based treatment. In some embodiments, PLC-based treatment can be administered before EV-based treatment. In some embodiments, PLC and EV are administered as a mixture. It is also contemplated to administer a mixture containing PLC and EV, followed by a treatment regimen that essentially includes EV or its derivatives or essentially includes PLC or its derivatives, depending on the patient's needs.

[0089] Some embodiments of the present disclosure provide compositions suitable for cryopreservation, including PLC or a derivative thereof or a lysate thereof, or PRP derived therefrom, or precursor cells that produce PLC or a derivative thereof or PRP derived therefrom, and a cryopreservative. 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, propylene glycol, albumin, or a combination thereof. In some embodiments, the cryopreservative is cellulose. Combinations comprising one or more cryopreservatives are also encompassed by the present disclosure. In some embodiments, the composition is frozen (i.e., the composition is stored at a temperature between about -80°C and -200°C). Cryoprotectants can be classified as permeating and non-permeating. Non-permeating cryoprotectants only alter the freezing properties of the extracellular medium, whereas permeating cryoprotectants can modify the composition of both the intracellular and extracellular media.

[0090] In a non-limiting embodiment, PLC or a derivative thereof or a lysate thereof, or PRP derived therefrom, or precursor cells that produce PLC or a derivative thereof or PRP derived therefrom, are enriched in growth factors or enriched in growth factors (Angiopoitin-1, bFGF, EGF, FGF, HGF, IGF-I, IGF-II, PDAF, PDEGF, PDGF). and VEGF), cytokines (e.g., IL-1B, IL-6, IL-8, IL-10, TNF-A, MIG, MCP-1, IP-10) or chemokines (e.g., ENA-78 (CXCL5), IL-8 (CXCL8), monocyte chemotactic protein, MCP-3 (CCL7), MIP-1A (CCL3), NAP-2 (CXCL7), PF4 (CXCL4), or inflammatory mediators (e.g., PGE2), macrophage inflammatory protein-1 (MIP-1), or activation regulator expressed and secreted by normal T cells (RANTES)) from a concentration of donor platelets. The concentration of growth factors or cytokines in PLC or its derivatives or lysates thereof, or PRP derived therefrom, or PLC or its derivatives (e.g., lysates form PLC or derivatives derived therefrom), or PRP enriched from precursor cells that produce PLC or its derivatives or PRP derived therefrom, can be diluted according to the needs of the subject. In some embodiments, PLC can 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 and 1B.

[0091] PLC or a derivative thereof or a lysate thereof, or PRP derived therefrom, or precursor cells that make PLC or a derivative thereof or PRP derived therefrom, or a combination thereof, can be administered or applied to damaged tissue of the skeletal musculature, for example, sprained ligaments (connecting bone to bone), strained or torn muscles (which may be torn), torn tendons, osteoarthritis, fractured bones (which may be cracked or shattered) or surrounding injured tissue, bone dislocation (i.e., bones within a joint may become completely separated from one another (called a dislocation) or only partially separated in position (called a subluxation)), sprains, strains, or other musculoskeletal injuries, completely or partially torn tendons, muscle spasms, nerve pain, whiplash, sports injuries, myocardial infarction or ischemia-related injuries (such as limb ischemia, lower limb ischemia, myocardial ischemia, organ ischemia, or ischemic heart disease), surgical procedures (which are difficult to heal), lung disease, heart disease.

[0092] Tissue damage or injury sites can be determined by well-established techniques, such as imaging studies, including but not limited to MRI, X-rays, and CT scans. X-rays can also be performed to check for fractures and dislocations. Furthermore, X-rays can show abnormalities in bone position, which may suggest sprains or other soft tissue injuries. Magnetic resonance imaging (MRI) can show soft tissues that are not normally visible on X-rays. MRI is therefore useful for detecting injuries to tendons, ligaments, cartilage, and muscles. Other techniques, such as, but not limited to, positron emission tomography (PET), single-photon emission computed tomography (SPECT), electrical impedance tomography (EIT), electrical source imaging (ESI), magnetic source imaging (MSI), laser optical imaging (NOGA) mapping, and ultrasound techniques, can also be applied to determine injury sites. A physician may examine the subject before performing one or more of the above tests.

[0093] PLC or its derivatives or their lysates, or PRP derived therefrom, or precursor cells or compositions thereof that produce PLC or its derivatives or PRP derived therefrom, can be administered via different routes (such as, but not limited to, systemic, local, etc.) using implantable devices (such as, but not limited to, stents, meshes (e.g., polymer meshes or bioabsorbable meshes), adhesive biomaterials (e.g., naturally occurring or synthetic biopolymers), or other devices known to those skilled in the art). For example, variables such as the appropriate timing, treatment cycle, location, and technique of knee injections (e.g., intravenous or intradermal injections) in patients undergoing knee osteoarthritis treatment can vary depending on whether PLC is administered once or multiple times. PLC or its derivatives can be administered daily, weekly (PLC injection once every three weeks), or monthly (two injections per month), with three injections at 15-day or 21-day intervals. Treatment regimens can vary depending on the needs of the patient. The location and technique for application of PLC or its derivatives or lysates or PRP derived therefrom (e.g., local injection into the knee) can be lateral, superolateral, parapatellar, mid-lateral patellar, etc. With each such treatment, one or more other therapeutic agents can be co-administered or administered at regular intervals from each other, as needed.

[0094] When a stent is used, it can be a closed-cell or open-cell stent, as is well known to those skilled in the art. PLC or its derivatives or lysates, or PRP derived therefrom, can be directly bonded to a metal stent or to a matrix polymer, which acts as a drug reservoir to ensure supply retention and uniform distribution on the stent during stent placement. The type, composition, and design of the polymer coated on the stent generally determine the elution rate for sustained release of PLC or its derivatives or lysates, or PRP derived therefrom, over weeks or months after in situ implantation. Coating materials can be classified as organic versus inorganic, biodegradable versus non-biodegradable, and synthetic versus naturally occurring.

[0095] In some embodiments, the composition comprising PLC or a derivative thereof or a lysate thereof or PRP derived therefrom, or precursor cells that produce PLC or a derivative thereof or PRP derived therefrom, comprises between 1% and 25% by weight, between 25% and 50% by weight, between 50% and 75% by weight, or between 75% and 100% by weight of PLC or a derivative thereof or platelet-rich plasma derived therefrom.

[0096] In some embodiments, the composition comprises between 1% and 25% by weight, between 25% and 50% by weight, between 50% and 75% by weight, or between 75% and 98% by weight of a bulking agent. In some embodiments, the composition comprises between 1% and 25% by weight, between 25% and 50% by weight, between 50% and 75% by weight, or between 75% and 98% by weight of at least one excipient or carrier. Thus, the weight percentages of PLC or its derivatives or platelet-rich plasma derived therefrom, bulking agents, excipients, or carriers can be adjusted once. For example, in an embodiment of the present disclosure, the composition may comprise a) between 1% and 50% by weight of PLC or its derivatives or lysates or platelet-rich plasma derived therefrom, b) between 1% and 25% by weight of a bulking agent, and / or c) between 50% and 98% by weight of at least one excipient or carrier.

[0097] In some embodiments, adhesive biopolymer materials may include, but are 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 PLC or its derivatives or lysates or PRP derived therefrom are contained in the adhesive biomaterial, the biological agent adheres to the target site to prolong the retention time of PLC or its derivatives or lysates or PRP derived therefrom in the lesion (e.g., ocular lesion) and improve the therapeutic effect of local diseases (e.g., dry eye disease). Higher local drug concentration at the absorption site and in 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 to facilitate the diffusion of PLC, its derivatives, its lysates, or PRP derived therefrom. Furthermore, PLC, its derivatives, its lysates, or PRP derived therefrom can adhere directly to the mucosa and be absorbed by mucosal tubules, increasing its bioavailability. Furthermore, adhesive biomaterials can be used to prepare controlled-release formulations of PLC, its derivatives, its lysates, or PRP derived therefrom, which can reduce the frequency of administration and improve the health of patients requiring PLC-based therapy.

[0098] In some embodiments, PLC or its derivatives or their lysates, or PRP derived therefrom, or precursor cells that produce PLC or its derivatives or PRP derived therefrom, can be used to treat dry eye disease. Dry eye disease is classified into two categories: (i) tear deficiency and (ii) evaporation. Causes of Sjögren's syndrome or non-Sjögren's syndrome include exocrine adenopathy, in which tear secretion deficiency occurs due to an autoimmune process affecting the lacrimal gland, salivary gland, and other organs of the body (Sjögren's syndrome), or non-Sjögren's syndrome, in which lacrimal gland disease or lacrimal gland obstruction and reflex changes occur without autoimmune factors playing a role. Some causes of Sjögren's syndrome or non-Sjögren's syndrome include age-related dry eye disease, congenital anlacrimation, familial autonomic neuropathy, sarcoidosis, lymphoma, AIDS (acquired immune deficiency syndrome), gland denervation, lacrimal gland obstruction such as pemphigus, trigeminal nerve injury, diabetes, neurotrophic keratopathy, contact lens use, and motor reflex blockage due to injury to the VII pair. Evaporative causes of dry eye disease are due to several factors, including oil deficiency, eyelid changes, contact lens use, or ocular surface diseases (such as allergic conjunctivitis), and iatrogenic dry eye disease that occurs after the use of systemic or topical medications or after surgical or non-surgical procedures.

[0099] PLC or its derivatives or lysates, PRP derived therefrom, or precursor cells that produce PLC or its derivatives or PRP derived therefrom can be applied in eye drops. For example, 2 to 3 milliliters of this concentrated solution can be included in a sterile eye drop. The eye drop can be kept at -20°C for long-term storage. When it is desired to use eye drops containing PLC or its derivatives or lysates, or PRP derived therefrom, the patient can thaw the eye drop and store it at +4°C. The patient can use these eye drops 1 to 6 times daily for one, two, three, or more months. In some embodiments, the patient can use the eye drop once daily for one, two, three, or four weeks or more.

[0100] In some embodiments, PLC or its derivatives or its lysate or PRP derived therefrom, or precursor cells that produce PLC or its derivatives or PRP derived therefrom can be injected or applied topically to skin regions such as the aging skin of the face, scalp, neck, chest, hand, arm, leg, abdomen or buttocks.It is expected that PLC or its derivatives or its lysate or PRP derived therefrom, or precursor cells that produce PLC or its derivatives or PRP derived therefrom can be applied to the scalp to extend the lifespan of hair follicles and increase hair growth.For use on the skin or scalp, PLC or its derivatives or its lysate or PRP derived therefrom, or precursor cells that produce PLC or its derivatives or PRP derived therefrom are provided in a composition comprising a cosmetically acceptable carrier. For example, in one aspect of the present disclosure, there is provided a method for generating hair follicles in the scalp or its bald area of ​​a subject, comprising contacting the scalp or its bald area with PLC or its derivative or its lysate or PRP derived therefrom, or precursor cells that produce PLC or its derivative or PRP derived therefrom.In addition, another precursor cell that can differentiate into hair follicle cells can be included together with the PLC or its derivative or its lysate or PRP derived therefrom of the present disclosure, or precursor cells that produce PLC or its derivative, so that when combined with the precursor cells that produce PLC or its derivative, the precursor cells can induce the differentiation of unrelated epidermal cells into hair follicle cells.In some embodiments, PLC or its derivative or its lysate or PRP derived therefrom, or precursor cells that produce PLC or its derivative or PRP derived therefrom (i.e., PRP source) are applied first, and then inducer cells are applied, or PRP or its source and inducer cells are applied simultaneously, or inducer cells are applied first, and then PRP or PRP source are applied. Except when applied simultaneously, one component may be applied within hours, days, or weeks of the other component, depending on the desired application needs of the subject.

[0101]

[0102] It may be desirable to administer other compounds to the patient, such as corticosteroids, tetrasubstituted pyrimidopyrimidines, glucocorticoids, beta-catenin proteins or polypeptides or agonists thereof, 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, meclofenamate sodium, meloxicam, oxaprozin, sulindac, and tolmetin), COX-2 inhibitors (e.g., rofecoxib, celecoxib, valdecoxib, and lumiracoxib), glucocorticoid receptor modulators, or DMARDs. The combination therapy of the present disclosure is particularly useful for treating immunoinflammatory disorders in combination with other agents (either biologics or small molecules) that modulate the immune response to positively impact the disease. Such agents include agents 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 enhance the action of anti-inflammatory cytokines (such as IL-10) and agents that inhibit the activity of pro-inflammatory cytokines (such as IL-6, IL-1, IL-2, IL-12, IL-15, or TNF-alpha). Agents that inhibit TNF-alpha include etanercept, adalimumab, infliximab, and CDP-870. In this example (an example of an agent that blocks the effects of TNF-alpha), the combination therapy reduces cytokine production, while etanercept or infliximab acts on the remaining portion of the inflammatory cytokine, enhancing treatment. Small molecule immunomodulatory agents include, for example, p38 MAP kinase inhibitors (such as VX702, SCIO469, doramapimod, RO30201195, SCIO323), TACE inhibitors (such as DPC333), ICE inhibitors (such as pranalcasan), and IMPDH inhibitors (such as mycophenolate and merimepodib).

[0103] Other therapeutic agents that may be administered before, simultaneously with, or at an interval after administration of PLC or its derivatives, lysates thereof, or PRP derived therefrom include thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates (such as busulfan, improsulfan, and piposulfan); aziridines (such as benzodopa, carboquone, meturedopa, and uredopa); ethyleneimines and methylamelamines (altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine); acetogenins (specifically butalasin and butalasinone); delta-9-tetrahydrocannabinol (dronabinol, Marinol™); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (synthetic analogs topotecan (Hycamtin™), CPT-11 (irinotecan, CAMPTOSAR™), acetylcamptothecin camptothecin, scopolecin, and 9-aminocamptothecin; bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and biceresin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictiin; spongistatin; nitrogenoma steroids (such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, fenesterine, prednimustine, trofosfamide, and uracil mustard); nitrosoureas (such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine); antibiotics (such as enediyne antibiotics, e.g., calicheamicin, specifically, calicheamicin gamma I and calicheamicin omega II);CDP323, an oral alpha-4 integrin inhibitor; dynemicins (including dynemicin A); esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, 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 D-99 (MYOCET™), pegylated liposomal doxorubicin (CAELYX™), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin (such as mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, and the like; antimetabolites (such as methotrexate, gemcitabine (GEMZAR™), tegafur (UFTORAL™), capecitabine (XELODA™), epothilones, and 5-fluorouracil; Fluorouracil (5-FU), etc.; combretastatins; folic acid analogs (denopterin, methotrexate, pteropterin, trimetrexate, etc.); purine analogs (fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc.); pyrimidine analogs (ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, etc.); androgens (calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.); corticosteroid inhibitors (aminoglutethimide, mitotane, trilostane, etc.); folic acid supplements (folinic acid, etc.); aceglatone;Aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids (such as maytansine and ansamitocin); mitoguazone; mitoxantrone; mopidammol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK™ polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicone; 2,2',2'-trichlorotriethylamine; trichothecenes (specifically, T-2 toxin, veraculin A, roridin A, and anguidine); urethane; vindesine (ELDISINE™, FILDESIN™); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabininoside ("Ara-C"); thiotepa; taxoids (e.g., paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, NJ), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE™, Rhome-Poultry Oncology, Princeton, NJ). Rorer, Antony, France); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents (such as cisplatin, oxaliplatin (e.g., ELOXATIN™), and carboplatin); vincas that prevent tubulin polymerization to form microtubules (including vinblastine (VELBAN™), vincristine (ONCOVIN™), vindesine (ELDISINE™, FILDESIN™), and vinorelbine (NAVELBINE™)); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin;ibandronate; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid (including bexarotene (TARGRETIN™)); bisphosphonates (clodronate (e.g., BONEFOS™ or OSTAC™), etidronate (DIDROCAL™), NE-58095, zoledronic acid / zoledronate (ZOMETA™), alendronate (FOSAMAX™), pamidronate (AREDIA™), tiludronate (S KELID™), or risedronate (ACTONEL™); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell growth (e.g., PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R) (e.g., erlotinib (Tarceva™)); and VEGF-A, which reduce cell proliferation); vaccines (THERATOPE™ vaccines and gene therapy) vaccines (e.g., ALLOVECTIN™ vaccine, LEUVECTIN™ vaccine, and VAXID™ vaccine); topoisomerase 1 inhibitors (e.g., LURTOTECAN™); rmRH (e.g., ABARELIX™); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT™, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib ( VELCADE™); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors (such as oblimersen sodium (GENASENSE™)); pixantrone; EGFR inhibitors; tyrosine kinase inhibitors; serine-threonine kinase inhibitors (such as rapamycin (sirolimus, RAPAMUNE™)); farnesyltransferase inhibitors (such as lonafarnib (SCH6636, SARASAR)); and pharmaceutically acceptable salts, acids, or derivatives of any of the above;and combinations of two or more of the above (such as CHOP (an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone); and FOLFOX (an abbreviation for a treatment regimen using oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin)), as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above;

[0104] Therapeutic agents as defined herein also include "antihormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block, or inhibit the effects of hormones that can promote cancer growth. Therapeutic agents include, but are not limited to, hormones themselves (antiestrogens and selective estrogen receptor modulators (SERMs) such as tamoxifen (including NOLVADWX™ tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and toremifene); aromatase inhibitors that inhibit the enzyme aromatase, which controls estrogen production in the adrenal glands (such as 4(5)-imidazole, aminoglutethimide, MEGASE™ megestrol acetate, AROMASIN™ exemestane, formestany, fadrozole, RIVISOR™ vorozole, FEMARA™ letrozole, and ARIMIDEX™ anastrozole); and antiandrogens (such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin). and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in aberrant cell growth (such as PKC-alpha, Raf, and H-Ras); ribozymes (such as VEGF expression inhibitors (e.g., ANGIOZYME™ ribozymes) 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 inhibitors; ABARELIX™ rmRH; vinorelbine and esperamicin, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above. Immunomodulatory drugs

[0105] In some embodiments, the present disclosure also encompasses immunomodulatory agents for use with the disclosed PLC or derivatives thereof or lysates thereof, or precursor cells that produce PRP from PLC or derivatives thereof, lysates thereof, or PRP-producing PLC or derivatives thereof, lysates thereof, or precursor cells derived therefrom. The term "immunomodulatory agent" refers to a class of drugs that modify immune system responses or immune system function, such as by stimulating antibody formation and / or inhibiting peripheral blood cell activity, and includes, but is not limited to, thalidomide (αN-phthalimido-glutarimide) and its analogs, REVLIMID™ (lenalidomide), ACTI-MID™ (pomalidomide), OTEZLA™ (apremilast), and pharmaceutically acceptable salts or acids thereof.

[0106] The treatment of the present disclosure can be performed alone or in combination with other treatments, and can be provided at home, in a doctor's office, a clinic, an outpatient department of a hospital, or in a hospital. If necessary, treatment can be started in a hospital, or can be started as an outpatient, so that a doctor can monitor the effect of treatment and make any necessary decisions. The duration of treatment depends on the type of disease or disorder being treated, the age and condition of the patient, the stage and type of the patient's disease, and how the patient responds to treatment. If necessary, people who are at a higher risk of developing inflammatory diseases (for example, people with age-related hormonal changes) can receive treatment to prevent or delay the onset of symptoms.

[0107] In combination therapy, the dosage and administration frequency of each component of the combination can be independently controlled.For example, one compound can be administered once, twice or three times a day, or once a week or once a month, while the second compound can be administered once a day, or once a week or once a month.Combination therapy can be administered in an on-off cycle, including a rest period, so that the patient's body has a chance to recover from any unforeseen side effects so far.Also, the compounds can be formulated together so that both compounds can be delivered in one administration.Except when administered simultaneously, one component in combination therapy can be administered within a few hours, days or weeks of the other component, depending on the desired administration needs of the subject.

[0108] In some embodiments, 10 6 PLCs or approximately 10 6 ~10 7 PLCs or approximately 10 7 ~10 8 PLCs or approximately 10 8 ~10 9 PLCs or approximately 10 9 ~10 10 PLCs or 10 10 More than about (1, 2, 3, 4, 5, 6, 7, 8, or 9) x 10 PLCs are administered to a subject. 6 ~(1, 2, 3, 4, 5, 6, 7, 8, or 9) x 10 7 PLCs, approximately (1, 2, 3, 4, 5, 6, 7, 8, or 9) x 10 7 PLCs ~ (1, 2, 3, 4, 5, 6, 7, 8, or 9) x 10 8 PLCs, or approximately (1, 2, 3, 4, 5, 6, 7, 8, or 9) x 10 8 PLCs ~ (1, 2, 3, 4, 5, 6, 7, 8, or 9) x 10 9 PLCs or approximately (1, 2, 3, 4, 5, 6, 7, 8, or 9) x 10 9 PLCs ~ (1, 2, 3, 4, 5, 6, 7, 8, or 9) x 10 10 PLCs may be administered to a subject.

[0109] The present disclosure provides kits comprising PLC or a derivative thereof or a lysate thereof, or PRP derived therefrom (i.e., a lysate derived from PLC or a derivative thereof) for use in accordance with the present disclosure, or precursor cells that produce PLC or a derivative thereof or PRP derived therefrom, and optionally a carrier, buffer, emulsifier, or excipient, the kit further comprising instructions for administration to a subject. The kit may optionally further comprise one or more drugs in one or more containers. The kit may further comprise a label or package insert. The package insert provides instructions regarding use, dosage, indications, administration, contraindications, and / or warnings regarding the use of such contents of the kit. Containers may include vials, bottles, syringes, blister packs, and the like. The active agent (e.g., PLC or any additional agent included in the kit) can typically be stored as a solid composition, a lyophilized formulation, or an aqueous solution.

[0110] The following examples are presented so as to fully disclose and describe to those of ordinary skill in the art how to make and use the assay, screening, and treatment methods of the present disclosure, and the examples are not intended to limit the scope of what the inventors regard as their disclosure. [Example]

[0111] Example Example 1: Osteoarthritis (OA) study of PLC in a rat MIA model showing significant improvement in OA symptoms A rat monoiodoacetate (MIA) osteoarthritis model was used in this study, as shown in Figure 2. MIA inhibits glyceraldehyde-3-phosphate, which causes chondrocyte death and leads to cartilage degeneration and subchondral bone lesions, similar to human OA disease.

[0112] Groups of 20 male Sprague Dawley rats received an intra-articular injection of 2 mg of MIA into the right knee on day 0. On day 18, rats were treated with a single dose of Plasmalyte buffer (negative control), PLC, PLC lysate, or human PRP by intra-articular injection into the right knee. Oral dexamethasone was used as a positive control, but this treatment was stopped early in the study because rats receiving this regimen began to lose weight.

[0113] Weight bearing was used to assess pain in these animals. Rats stood while balancing on their left hind paw (RL) and right hind paw (RR). Healthy rats balanced their weight equally on both legs, while osteoarthritic rats balanced more weight on the healthy limb (RL) compared to the affected limb (RR). Successful PLC treatment resulted in the cessation of pain and a reduction in the weight bearing difference over time. Weight bearing was tested 2 weeks (Figures 3A-3B), 4 weeks (Figures 3C-3D), and 8 weeks (Figures 3E-3F) after treatment. At 8 weeks, PLC-treated rats showed significant improvement in OA symptoms. Successful PLC treatment resulted in the cessation of pain in the affected limb and a reduction in the weight bearing difference over time. Example 2 PLC-based diabetic wound healing.

[0114] Experimental procedure.

[0115] Animals: Species: rat; strain: ZDF (ZDF-Leprfa / Crl, obese), lean control rats; animal source: Charles River Lab; age or weight: 16 weeks; sex: male.

[0116] Randomization: Baseline blood glucose and body weight are measured. Animals are assigned to treatment groups based on these parameters. Rats with blood glucose above 14 mmol / L or 252 mg / dl are used in the study.

[0117] Study design: Study duration: 15 days; Number of groups: 2; Number of animals per group: 10; Total number of animals: 20.

[0118] Methods: Confirmation of Diabetes: Male obese catheterized ZDF rats were obtained from Charles River Lab. All rats were housed singly. Diabetic status was confirmed by assessing blood glucose from a tail snip. Blood glucose was measured using a Glucocard Vital blood glucose meter (Arkray, Minneapolis, MN) and levels were reported as mg / dL. The blood glucose meter was calibrated before each study. Blood (less than 5 μL) was collected from a tail snip and applied directly to a glucose test strip. Rats with glucose levels ≥ 14 mmol / L were enrolled in the study.

[0119] Wound induction, treatment, and post-wound monitoring: Animals are shaved and prepped, and 10 mm wounds (bilaterally) are created by punch biopsy on the dorsal skin toward the scapular region. After excision wounding, the wounds are treated with PLC or PLC covered with Tegaderm™ (3M) dressing. The wound dressing is replaced on wound measurement days (i.e., days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, and 14). Wound size is measured using digital calipers (mm) immediately after wound induction on day 0 and on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, and 14. Wound closure rate is also calculated. Rats are anesthetized to allow for measurements. PLC is expected to shorten wound healing time in these rats. Example 3: PLC-induced hair growth

[0120] The animals were anesthetized with isoflurane and their backs were shaved using an electric razor to remove most of the hair in a single location, avoiding cutting or nicking the skin. 2The area is shaved. Hair is removed until skin color is visible. Animals are evaluated to determine if there is pigmentation underneath the hair. If there is pigmentation, scars from cuts, amputations, or fighting, the animals are removed from the study. Baseline photography and scoring for each rat is performed after shaving, and animals are randomized based on skin color and initial BW. Hair growth scores are taken twice a week (usually Monday and Friday), and animals are weighed and photographed weekly throughout the course of the study.

[0121] According to the study design (Table 1), vehicle and test substances are administered subcutaneously every three days in the shaved area, while minoxidil (positive control) is applied topically once daily. For topical application, 100 ul of solution is used for each application. For SC application, the test substance is injected (100 ul) into the observation site. From these studies, it is predicted that PLC will facilitate hair growth. Table 1: [Table 1]

[0122] For hair growth assessment, rats were scored based on hair growth using the following scale: [Table 2]

[0123] From the foregoing description, it will be apparent that the embodiments of the present disclosure can be varied and modified to adapt to various applications and conditions. Such embodiments are within the scope of the following claims. Any definition of a variable herein recites a list of elements, and such definition of the variable includes any single element or combination (or subcombination) of the listed elements. Any embodiment described herein includes any single embodiment or combination or portion thereof with any other embodiment.

[0124] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference. The present invention provides, for example, the following items. (Item 1) 1. A method of treating, repairing, or ameliorating a symptom in a subject in need thereof, the method comprising administering to the subject a treatment composition comprising an effective amount of platelet-like cells (PLCs) or derivatives thereof, wherein the symptom is one or more of disease, damaged tissue, hair loss, wound healing, a wound healing-related disorder, or skin damage or aging. (Item 2) 10. The method of claim 1, wherein the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, an anti-hormonal agent, or an immunomodulatory agent, or a combination thereof. (Item 3) 2. The method of claim 1, wherein the composition further comprises platelet-rich plasma (PRP) derived from the subject. (Item 4) 4. The method according to any one of items 1 to 3, wherein the composition is diluted to a physiological concentration in a carrier, the carrier comprising a diluent or excipient. (Item 5) The method according to item 4, wherein the carrier is plasma, a plasma substitute, or Plasmalyte. (Item 6) Item 7. The method of any one of items 1 to 3, wherein the composition is administered to treat one or more of the following conditions: osteoarthritis, damaged tissue, tendon injury, ligament injury, bone repair, wound healing or wound healing-related disorders, dry eye disease, alopecia, or skin aging or damage. 7. The method of claim 6, wherein the damaged tissue comprises damage to the skeletal musculature. (Item 8) 7. The method of claim 6, wherein the skin aging or damage is present on the face, scalp, neck, chest, hands, arms, legs, abdomen, or buttocks of the subject, or a combination thereof. (Item 9) Item 7. The method according to item 6, wherein the dry eye disease is caused by Sjogren's syndrome or non-Sjogren's syndrome. (Item 10) 4. The method according to any one of items 1 to 3, wherein said PLC or derivative thereof does not contain red blood cells or hemoglobin content or white blood cells. (Item 11) 4. The method of any one of items 1 to 3, wherein the composition further comprises extracellular vesicles (EVs). (Item 12) 4. The method of any one of items 1 to 3, wherein the composition is formulated for application to an injury site for therapeutic use. (Item 13) 3. The method of claim 2, wherein the composition further comprises another therapeutic agent. (Item 14) 13. The method of claim 12, wherein the formulation is in a buffer, diluent, or excipient, or a combination thereof. (Item 15) 4. The method according to any one of items 1 to 3, wherein the composition is lyophilized. (Item 16) 4. The method according to any one of items 1 to 3, wherein the composition is implanted in an implantable device. (Item 17) 4. The method according to any one of items 1 to 3, wherein the composition is cryopreserved. (Item 18) 4. The method of any one of items 1 to 3, wherein the composition is administered locally at or near the site of injury or disease. (Item 19) 1. A method for producing PLC containing PRP, comprising: culturing the population of progenitor cells ex-vivo in a bioreactor for a period of time during which said progenitor cells differentiate into mature megakaryocytes; isolating a population of PLCs or derivatives thereof that have been subjected to separation from said megakaryocytes by a pressure gradient in said bioreactor; concentrating the PLC or its derivatives; optionally dissolving the PLC or derivative thereof; and Mixing with donor-derived platelet-rich plasma A method comprising: (Item 20) The progenitor cells are selected from the group consisting of human induced pluripotent stem cells (iPSCs), hematopoietic stem cells, and CD34 + Umbilical cord blood stem cells (UCB cells), CD34 + 21. The method of claim 19, wherein the progenitor cells are selected from one or more of mobilized peripheral blood cells (MPB cells), or CD34+ bone marrow cells. 20. A method for treating a subject in need thereof, further comprising administering to the subject an effective amount of PLC or a derivative thereof according to item 19, or PRP derived therefrom. (Item 22) 22. The method of claim 21, further comprising another therapeutic agent. (Item 23) A method of treating a patient with an isolated in vitro non-nuclear population of platelet-like cells (PLC) or derivatives thereof, wherein said non-nuclear population exhibits the following characteristics: i) is derived from the reprogramming of somatic cells, progenitor cells, or stem cells, the products of which are passed through a bioreactor; ii) is not a cancerous cell; iii) does not exhibit uncontrolled proliferation or tumor formation in vivo, and wherein said PLC or derivatives thereof are administered in a therapeutic amount to said patient in need of such treatment. (Item 24) 24. The method according to item 23, further comprising the step of administering to the patient an extracellular vehicle (EV) or a derivative thereof, wherein the EV or derivative thereof is produced in a mixture comprising PLC or a PLC derivative. (Item 25) 25. The method of any one of items 23 to 24, wherein the disorder or injury is osteoarthritis, damaged tissue, tendon injury, ligament injury, bone repair, wound healing or wound healing-related disorder, dry eye disease, alopecia, or skin aging. (Item 26) A method for treating a disease or disorder associated with injury, wherein the injury comprises one or more of joint injury, tendon injury, arthritis, osteochondral lesion, tenosynovitis, bursitis, and ligament injury in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of PLC or a derivative thereof, wherein the composition is administered in a therapeutic amount, and a statistically significant reduction in injury recovery is measured after administration of the composition, and / or a statistically significant improvement in pain, stiffness, and function is measured after administration of the composition. (Item 27) 27. The method of claim 26, wherein the disorder or injury is osteoarthritis, damaged tissue, tendon injury, ligament injury, bone repair, wound healing or wound healing-related disorder, dry eye disease, alopecia, or skin aging. (Item 28) 28. The method of claim 27, wherein the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, an anti-hormonal agent, or an immunomodulatory agent, or a combination thereof. (Item 29) 29. The method of item 28, wherein the tissue regeneration agent is a growth factor selected from one or more growth factors selected from 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. (Item 30) 30. The method according to any one of items 26 to 29, wherein the composition is in the form of a granule, a tablet, a suspension in a liquid carrier, a capsule, or a powder. (Item 31) 30. The method of any one of items 26 to 29, wherein the composition comprises a) between 1% and 100% by weight of PLC or a derivative thereof or platelet-rich plasma derived therefrom, b) between 0% and 90% by weight of a bulking agent, and / or c) between 0% and 90% by weight of at least one excipient or carrier, and optionally d) platelet-rich plasma derived from the subject. (Item 32) 30. The method of any one of items 26 to 29, wherein the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, an anti-hormonal agent, or an immunomodulatory agent, or a combination thereof. (Item 33) A method for treating, repairing, or ameliorating disease, damaged tissue, hair loss, wound healing, or skin damage or aging in a subject in need thereof, comprising administering to the subject more than one dose of a treatment composition comprising an effective amount of platelet-like cells (PLCs) or derivatives thereof. (Item 34) 34. The method of claim 33, wherein the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, an anti-hormonal agent, or an immunomodulatory agent, or a combination thereof. (Item 35) 34. The method of item 33, wherein the more than one dose is administered daily, weekly, every two weeks, every three weeks, or monthly. (Item 36) 36. The method of any one of items 33 to 35, wherein the route of administration is topical, transdermal, or systemic. (Item 37) 36. The method according to any one of items 33 to 35, wherein the route of administration is intravenous, intraarterial, intramuscular, subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal, intraarticular, ocular, otic, or oral. (Item 38) A method for treating, repairing, or ameliorating disease, damaged tissue, hair loss, wound healing, or skin damage or aging in a subject in need thereof, comprising administering to the subject more than one dose of a treatment composition comprising an effective amount of platelet-like cells (PLCs) or derivatives thereof, wherein the disease is not cancer or the treatment is not for cancer. (Item 39) 37. The method of items 1, 19, 23, 26, 32, or 36, wherein the PLC or derivative thereof is produced in a fluidic device or a bioreactor. (Item 40) A composition for treating a symptom in a subject, produced according to the method of any one of items 1 to 39. (Item 41) A composition for treating a symptom in a subject, comprising platelet-like cells (PLC) or derivatives thereof and platelet-rich plasma (PRP) derived from said subject. (Item 42) Item 43. The composition of item 41, wherein the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, an anti-hormonal agent, or an immunomodulatory agent, or a combination thereof. 42. The composition of claim 41, wherein the composition is diluted to a physiological concentration in a carrier, the carrier comprising a diluent or excipient. (Item 44) 42. The composition of claim 41, wherein the carrier is plasma or a plasma substitute or Plasmalyte. (Item 45) 42. The composition of claim 41, wherein said PLC or derivative thereof does not contain red blood cells or hemoglobin content or white blood cells. (Item 46) 42. The composition of claim 41, wherein the composition further comprises extracellular vesicles (EVs). (Item 47) 42. The composition of claim 41, wherein the composition is formulated for application to a site of injury for therapeutic use. (Item 48) 42. The composition of claim 41, wherein the composition further comprises another therapeutic agent.

Claims

1. Use of a composition in the manufacture of a medicament for treating a condition in a subject, said composition comprising a population of precursor cells that give rise to platelet-like cells (PLCs) or derivatives thereof, wherein said population of precursor cells that give rise to said PLCs or derivatives thereof comprises one or more of the following factors compared to reference bone marrow-derived megakaryocytes or platelets: 50% more vascular endothelial growth factor (VEGF)-A, 50% more AP-2, 50% more IL-8, 50% more endoglin, 50% more VEGF-D, 50% more endothelin, 50% more FS, 50% more epidermal growth factor (EGF), 50% more TNF-alpha, 50% less platelet-derived growth factor (PDGF) BB, 50% less VEGF-C, or 50% less HB-EGF.

2. The use of claim 1, wherein the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, an anti-hormonal agent, an immunomodulatory agent, extracellular vesicles (EVs) or another therapeutic agent, or a combination thereof.

3. The use described in claim 1, wherein the composition is diluted to a physiological concentration in a carrier, and the carrier comprises a diluent or excipient.

4. The use described in claim 3, wherein the carrier is plasma or a plasma substitute or a balanced crystalloid solution that mimics human plasma.

5. The use described in claim 1, wherein the composition is administered to treat osteoarthritis, wound healing or wound healing-related disorders, or dry eye disease.

6. The use of claim 5, wherein the composition is administered to treat osteoarthritis.

7. The use described in claim 5, wherein the composition is administered to treat dry eye disease.

8. The use of claim 1, wherein the precursor cells from which the PLC or its derivatives are produced do not contain red blood cells or hemoglobin content or white blood cells.

9. The use described in claim 1, wherein the composition further comprises extracellular vesicles (EVs) in the range of 65 nm to 10 μm.

10. The use described in claim 1, wherein the precursor cells that produce the PLC or its derivatives are generated by exposure to one or more of shear stress, mechanical strain, or a pulsed electromagnetic field.

11. The use of claim 1, wherein the composition is one or more of: formulated for application to the site of injury for therapeutic use; lyophilized; implanted in an implantable device; cryopreserved; or administered locally at or near the site of injury or disease.

12. The use of claim 11, wherein the formulation is carried out in a buffer, diluent, or excipient, or a combination thereof.

13. Use of a composition in the manufacture of a medicament for treating a disease, disorder, or injury, wherein the composition comprises a population of precursor cells that produce platelet-like cells (PLCs) or derivatives thereof, wherein the composition is administered in a therapeutic amount, and a reduction in injury recovery time is measured after administration of the composition, and / or an improvement in pain, stiffness, and function is measured after administration of the composition; The use wherein the population of precursor cells from which the PLC or derivative thereof is produced comprises one or more of the following factors compared to reference bone marrow-derived megakaryocytes or platelets: 50% more vascular endothelial growth factor (VEGF)-A, 50% more AP-2, 50% more IL-8, 50% more endoglin, 50% more VEGF-D, 50% more endothelin, 50% more FS, 50% more epidermal growth factor (EGF), 50% more TNF-alpha, 50% less platelet-derived growth factor (PDGF) BB, 50% less VEGF-C, or 50% less HB-EGF.

14. The use of claim 13, wherein the disease, disorder, or injury is osteoarthritis, damaged tissue, tendon injury, ligament injury, bone repair, wound healing or wound healing-related disorder, dry eye disease, alopecia, or skin aging.

15. The use of claim 13, wherein the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, an anti-hormonal agent, an immunomodulatory agent, or a combination thereof.

16. The use of claim 15, wherein the tissue regeneration agent is a growth factor selected from one or more of transforming growth factor (TGF), fibroblast growth factor (FGF), PDGF, EGF, 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), and combinations thereof.

17. The use of claim 13, wherein the composition is in the form of a gel, ointment, granules, tablets, a suspension in a liquid carrier, capsules, or powder.

18. The use described in claim 13, wherein the composition comprises: a) between 1% and 100% by weight of precursor cells that produce the PLC or a derivative thereof or platelet-rich plasma derived therefrom; b) between 0% and 90% by weight of a bulking agent; c) between 0% and 90% by weight of at least one excipient or carrier; and optionally d) platelet-rich plasma derived from a subject.

19. Use of a composition in the manufacture of a medicament for treating dry eye disease, osteoarthritis, and wound healing or wound healing related disorders in a subject in need of such treatment, wherein the composition comprises a population of precursor cells that produce platelet-like cells (PLCs) or derivatives thereof, The use wherein the population of precursor cells from which the PLC or derivative thereof is produced comprises one or more of the following factors compared to reference bone marrow-derived megakaryocytes or platelets: 50% more vascular endothelial growth factor (VEGF)-A, 50% more AP-2, 50% more IL-8, 50% more endoglin, 50% more VEGF-D, 50% more endothelin, 50% more FS, 50% more epidermal growth factor (EGF), 50% more TNF-alpha, 50% less platelet-derived growth factor (PDGF) BB, 50% less VEGF-C, or 50% less HB-EGF.

20. The use of claim 19, wherein the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, an anti-hormonal agent, or an immunomodulatory agent, or a combination thereof.

21. The use of claim 19, wherein the composition is administered more than once a day, daily, weekly, every two weeks, every three weeks, or monthly.

22. The use of claim 19, wherein the route of administration is topical, transdermal, systemic, intravenous, intraarterial, intramuscular, subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal, intraarticular, ocular, otic, or oral.

23. A composition for treating a condition in a subject, said composition comprising a population of precursor cells that give rise to platelet-like cells (PLCs) or derivatives thereof, wherein said population of precursor cells that give rise to said PLCs or derivatives thereof comprises one or more of the following factors compared to reference bone marrow-derived megakaryocytes or platelets: 50% more vascular endothelial growth factor (VEGF)-A, 50% more AP-2, 50% more IL-8, 50% more endoglin, 50% more VEGF-D, 50% more endothelin, 50% more FS, 50% more epidermal growth factor (EGF), 50% more TNF-alpha, 50% less platelet-derived growth factor (PDGF) BB, 50% less VEGF-C, or 50% less HB-EGF.

24. The composition of claim 23, further comprising a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, an anti-hormonal agent, an immunomodulatory agent, extracellular vesicles (EVs) or another therapeutic agent, or a combination thereof.

25. The composition of claim 23, wherein the composition is diluted to a physiological concentration in a carrier, the carrier comprising a diluent or excipient.

26. The composition described in claim 25, wherein the carrier is plasma or a plasma substitute or a balanced crystalloid solution that mimics human plasma.

27. ​​The composition described in claim 23, characterized in that the composition is administered to treat osteoarthritis, wound healing or wound healing-related disorders, or dry eye disease.

28. The composition of claim 27, wherein the composition is administered to treat osteoarthritis.

29. The composition described in claim 27, characterized in that the composition is administered to treat dry eye disease.

30. The composition of claim 23, wherein the precursor cells from which the PLC or its derivatives are produced do not contain red blood cells or hemoglobin content or white blood cells.

31. The composition described in claim 23, further comprising extracellular vesicles (EVs) in the range of 65 nm to 10 μm.

32. The composition of claim 23, wherein the precursor cells that produce the PLC or its derivatives are produced by exposure to one or more of shear stress, mechanical strain, or a pulsed electromagnetic field.

33. The composition of claim 23, wherein the composition is characterized in that it is one or more of: formulated for application to the site of injury for therapeutic use; lyophilized; implanted in an implantable device; cryopreserved; or administered locally at or near the site of injury or disease.

34. The composition of claim 33, wherein the formulation is carried out in a buffer, diluent, or excipient, or a combination thereof.

35. A composition for treating a disease, disorder, or injury, said composition comprising a population of precursor cells that produce platelet-like cells (PLCs) or derivatives thereof, characterized in that said composition is administered in a therapeutic amount, and a reduction in injury recovery time is measured after administration of said composition, and / or an improvement in pain, stiffness, and function is measured after administration of said composition; A composition wherein the population of precursor cells that produce the PLC or derivative thereof comprises one or more of the following factors compared to reference bone marrow-derived megakaryocytes or platelets: 50% more vascular endothelial growth factor (VEGF)-A, 50% more AP-2, 50% more IL-8, 50% more endoglin, 50% more VEGF-D, 50% more endothelin, 50% more FS, 50% more epidermal growth factor (EGF), 50% more TNF-alpha, 50% less platelet-derived growth factor (PDGF) BB, 50% less VEGF-C, or 50% less HB-EGF.

36. The composition of claim 35, wherein the disease, disorder, or injury is osteoarthritis, damaged tissue, tendon injury, ligament injury, bone repair, wound healing or wound healing-related disorder, dry eye disease, alopecia, or skin aging.

37. The composition of claim 35, wherein the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, an anti-hormonal agent, an immunomodulatory agent, or a combination thereof.

38. The composition of claim 37, wherein the tissue regeneration agent is a growth factor selected from one or more of transforming growth factor (TGF), fibroblast growth factor (FGF), PDGF, EGF, 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), and combinations thereof.

39. The composition of claim 35, wherein the composition is in the form of a gel, ointment, granules, tablets, a suspension in a liquid carrier, a capsule, or a powder.

40. The composition described in claim 35, wherein the composition comprises: a) between 1% and 100% by weight of precursor cells that produce the PLC or a derivative thereof or platelet-rich plasma derived therefrom; b) between 0% and 90% by weight of a bulking agent; c) between 0% and 90% by weight of at least one excipient or carrier; and optionally d) platelet-rich plasma derived from a subject.

41. A composition for treating dry eye disease, osteoarthritis, and wound healing or wound healing-related disorders in a subject in need thereof, the composition comprising a population of precursor cells that produce platelet-like cells (PLCs) or derivatives thereof; A composition wherein the population of precursor cells that produce the PLC or derivative thereof comprises one or more of the following factors compared to reference bone marrow-derived megakaryocytes or platelets: 50% more vascular endothelial growth factor (VEGF)-A, 50% more AP-2, 50% more IL-8, 50% more endoglin, 50% more VEGF-D, 50% more endothelin, 50% more FS, 50% more epidermal growth factor (EGF), 50% more TNF-alpha, 50% less platelet-derived growth factor (PDGF) BB, 50% less VEGF-C, or 50% less HB-EGF.

42. The composition of claim 41, wherein the composition further comprises a wound healing agent, a tissue regeneration agent, an anti-apoptotic agent, an anti-inflammatory agent, an anti-hormonal agent, or an immunomodulatory agent, or a combination thereof.

43. The composition of claim 41, wherein the composition is administered more than once a day, daily, weekly, every two weeks, every three weeks, or monthly.

44. The composition of claim 41, wherein the route of administration is topical, transdermal, systemic, intravenous, intraarterial, intramuscular, subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal, intraarticular, ocular, otic, or oral.

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