Ipsc-derived Anti-inflammatory macrophages and uses in modulation of inflammation and treatment of post-traumatic osteoarthritis

Abstract

Methods are provided for scalable differentiation and generation of chemically defined, functional human induced pluripotent stem cell (iPSC)–derived M2-like anti-inflammatory macrophages (iMac-M2), which exhibit stable expression of CD163 and CD206, robust phagocytic activity, and maintain anti-inflammatory phenotype under osteoarthritis-relevant inflammatory conditions. In co-culture with human osteoarthritic chondrocytes and synoviocytes, iMac-M2 significantly reduced the production of pro-inflammatory cytokines and matrix-degrading enzymes. In a rat destabilized medial meniscus model, a single intra-articular injection of iMac-M2 administered one week after injury restored gait symmetry, improved weight-bearing capacity by 42% at 16 weeks, preserved subchondral bone architecture, and reduced cartilage degeneration. The treatment modulated pain-related pathways as well as key inflammatory networks, without evidence of systemic immune activation or ectopic tissue formation. Uses of iMac-M2 are provided including for modulating inflammation, preserving joint integrity, and alleviating pain in post-traumatic osteoarthritis.

Description

IPSC-DERIVED ANTI-INFLAMMATORY MACROPHAGES AND USES IN MODULATION OF INFLAMMATION AND TREATMENT OF POST-TRAUMATIC OSTEOARTHRITISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application includes a claim of priority under 35 U.S.C. §119(e) to U.S. provisional patent application No. 63 / 745,418, filed January 15, 2025, the entirety of which is hereby incorporated by reference.FIELD OF INVENTION

[0002] This invention relates to cell therapies to treat joint injury and modulate inflammation in post-traumatic osteoarthritis.BACKGROUND

[0003] Post-traumatic osteoarthritis (PTOA) is a common and debilitating outcome of joint injuries, leading to chronic pain, functional disability, and substantial economic burden due to its high prevalence and costly management. Approximately 12% of all osteoarthritis cases are attributed to previous joint trauma, including fractures, ligament tears such as anterior cruciate ligament (ACL) ruptures and meniscal damage. Joint trauma initiates a cascade of biomechanical disruption and inflammatory signaling that progressively compromises joint integrity, frequently culminating in PTOA. Notably, up to 50% of joint injuries progress to PTOA, typically manifesting within 7-10 years after the initial trauma.

[0004] Unlike primary osteoarthritis, which predominantly affects older individuals, PTOA impacts patients across all age groups, with young adults and athletes being particularly vulnerable due to their high exposure to joint injuries. Recent studies underscore the alarming rise in PTOA prevalence, with projections showing it could affect as many as 40.6% of individuals by 2030, up from 21.1% in 2022. These trends highlight the clinical importance of early interventions aimed at altering disease trajectory rather than managing end-stage degeneration.

[0005] PTOA is now recognized as a whole-joint disease involving coordinated pathological changes in cartilage, subchondral bone, synovium, and periarticular tissues. Following joint injury, an acute inflammatory response is triggered, characterized by elevated levels of pro-inflammatory cytokines such as IL-ip, IL-6, IL-8, and TNF-a in the synovial fluid. While this response is initially required for tissue repair, its persistence promotes matrix metalloproteinase (MMPs) activation and cartilage degradation.4899-6723-7253 2 Page 1 of 52 065472-001005WOPT

[0006] Importantly, a subset of patients exhibits a dysregulated inflammatory response following ACL injury, marked by sustained elevation of pro-inflammatory mediators and cartilage degradation markers. Such unresolved inflammation is a major risk factor for PTOA development.

[0007] Although surgical techniques, such as ligament reconstruction, can restore joint stability, their impact on PTOA prevention remains limited. This limitation underscores a critical gap between biomechanical correction and long-term biological joint protection, indicating that mechanical restoration alone is insufficient to prevent PTOA.

[0008] Blunt cartilage compression during ACL injuries induces bone bruises and osteochondral microtrauma, associated with inflammatory cytokine dysregulation and early cartilage injury. These events initiate prolonged inflammatory cascades, with pathways such as IL-1 signaling playing a key role in PTOA onset and progression. Similarly, displaced intraarticular fractures exacerbate disease development by activating inflammatory pathways and promoting chondrocyte death, accelerating cartilage destruction. Mechanical loading further amplifies these effects by altering matrix metabolism and enhancing the production of inflammatory mediators, including nitric oxide and prostaglandin E2. While surgical interventions such as ACL repair restore partial mechanical stability, they frequently fail to resolve these inflammatory and catabolic processes, thereby limiting their ability to prevent PTOA progression. Bone bruises and osteochondral damage, commonly observed after acute joint trauma, illustrate the tight interplay between biomechanical injury and inflammation-driven cartilage degeneration, although their precise contribution to long-term functional outcomes remains under investigation.

[0009] Therefore, it is an object of the present disclosure to provide therapies for uses in restoring joint balance, protecting cartilage, and relieving pain after traumatic joint injury.

[0010] It is another object of the present disclosure to provide cell therapy and administration methods to intervene at an early, disease-modifying stage of PTOA.

[0011] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.4899-6723-7253 2 Page 2 of 52 065472-001005 WOPTSUMMARY OF THE INVENTION

[0012] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0013] Various embodiments provide methods for reducing inflammation in joint, treating joint injury, and / or inhibiting post-traumatic osteoarthritis in an individual in need thereof, which include administering to the individual a quantity of M2 phenotype-polarized macrophages derived from pluripotent stem cells. Various embodiments provide methods for administering anti-inflammatory macrophages to an individual in need thereof, which include administering a quantity of M2 phenotype-polarized macrophages derived from pluripotent stem cells. In preferably embodiments, the M2 phenotype-polarized macrophages are derived from induced pluripotent stem cells (iMac-M2). For administration in a human individual, the M2 phenotype-polarized macrophages are preferably derived from human induced pluripotent stem cells.

[0014] In various aspects, the iMac-M2 express CD68 and CD 163 for at least 34 days, at least 19 days, at least 16 days, at least 13 days, or at least 7 days after derivation from the iPSC. In various aspects, the iMac-M2 express CD14, CD68 and CD163 for at least 34 days, at least 19 days, at least 16 days, at least 13 days, or at least 7 days after derivation from the iPSC.

[0015] In various aspects, the iMac-M2 reduces IL6, ILip, and / or IL8 levels in the joint or in inflamed chondrocytes and / or inflamed synoviocytes. In various aspects, the iMac-M2 reduces ADAMTS5, MMP3, MMP9, and / or MMP13 levels in the joint or in inflamed chondrocytes and / or inflamed synoviocytes. Inflamed chondrocytes or inflamed synoviocytes may be chondrocytes or synoviocytes obtained from a patient with osteoarthritis. Alternatively, inflamed chondrocytes or inflamed synoviocytes are chondrocytes or synoviocytes having been exposed to inflammatory IL-ip or lipopolysaccharide (LPS), to induce an inflammatory state.

[0016] In some embodiments, the iMac-M2 are administered locally to the joint or o a site of osteoarthritis in the individual. In some embodiments, the administration of the iMac-M2 does not lead to an increase in blood IgM level. In some embodiments, the blood IgM level is not increased by more than 1%, 2%, 5%, 10%, or 20% as compared to pre-administration level.

[0017] In some embodiments, an individual in need of the administration is one who has had a joint injury or who is undergoing a joint injury. In some embodiments, an individual in need of the administration is one who is at risk of developing post-traumatic osteoarthritis.4899-6723-7253 2 Page 3 of 52 065472-001005WOPTIn some embodiments, an individual in need of the administration is one whose joint has inflammation or elevated levels of inflammatory markers. In some embodiments, an individual in need of the administration is one with a joint injury or inflamed joint or swelling / pain in the joint, but who does not have degenerative arthritis. In other embodiments, an individual in need of the administration is one who has degenerative arthritis. In some embodiments, the iMac-M2 are administered before osteoarthritis is diagnosed or developed in the individual. In some embodiments, the iMac-M2 are administered to an individual with a recent joint injury from 10 days or fewer than 10 days ago.

[0018] Exemplary joints for the administration or treatment of local pain, swelling or inflammation include a knee joint, a finger joint, a toe joint, a wrist joint, an ankle joint, an elbow joint, a hip joint, a should joint, a pivot joint, a joint between the vertebrae, or a joint where a thumb joins a hand. In some embodiments, the joint comprises a knee joint. In some embodiments, the joint comprises a hip joint. In some embodiments, the joint comprises an elbow joint.

[0019] In various embodiments, the iMac-M2 are produced by a process comprising: incubating isolated mononuclear phagocytes derived from pluripotent stem cells with a first culture media comprising macrophage colony-stimulating factor (M-CSF), wherein the mononuclear phagocytes derived from pluripotent stem cells differentiate into cells of macrophage lineage which express CD68; and incubating the cells of the macrophage lineage with a second culture media comprising M-CSF, IL-4, IL-6, and IL-13, wherein the cells of the macrophage lineage is polarized to an M2 phenotype and co-express CD 163 and CD206; thereby obtaining the cells of the macrophages lineage polarized to the M2 phenotype. In some embodiments, the M-CSF is at about 5-50 ng / mL or about 20 ng / mL. In some embodiments, the M-CSF / IL-4 / IL-6 / IL-13 combination contains each agent at about 5-50 ng / mL or about 20 ng / mL. In some embodiments, the incubating with the media containing M-CSF is for about 5 days or 4-7 days. In some embodiments the incubating with the media containing the M-CSF / IL4 / IL-6 / IL-13 combination is for about 2 days or 1-5 days.

[0020] In some embodiments, mononuclear phagocytes are derived from pluripotent stem cells in a process comprising: incubating induced pluripotent stem cells with a culture media comprising bone morphogenetic protein (BMP-4) to obtain cells in the form of embryoid bodies; incubating the cells of the form of embryoid bodies with a serum-free culture media comprising basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stem cell factor (SCF); followed by incubating with a second serum-free culture media comprising SCF, IL-3, thrombopoietin (TPO), macrophage colony-stimulating factor (M-4899-6723-7253 2 Page 4 of 52 065472-001005WOPTCSF), and Fms-like tyrosine kinase 3 ligand (FLT3 ligand); followed by incubating with a third serum-free culture media comprising FLT3 ligand, GM-CSF, and M-CSF, thereby obtaining mononuclear phagocytes derived from pluripotent stem cell, which express CD14. In some embodiments, the BMP4 is at about 60-100 ng / mL or about 80 ng / mL. In some embodiments, the bFGF / SCF / VEGF combination contains bFGF at about 20-30 ng / mL or about 25 ng / mL, SCF at about 80-120 ng / mL or about 100 ng / mL, and VEGF at about 60-100 ng / mL or 80 ng / mL. In some embodiments, the SCF / IL-3 / TPO / M-CSF / FLT3 ligand combination contains SCF at about 40-60 ng / mL or about 50 ng / mL, IL-3 at about 10-60 ng / mL or at about 20 ng / mL, TPO at about 1-10 ng / mL or about 5 ng / mL, M-CSF at about 40-60 ng / mL or about 50 ng / mL, and FLT3 ligand at about 40-60 ng / mL or about 50 ng / mL. In some embodiments, the M-CSF / FLT3 ligand / GM-CSF combination contains M-CSF at about 40-60 ng / mL or about 50 ng / mL, FLT3 ligand at about 40-60 ng / mL or about 50 ng / mL, and GM-CSF at about 20-30 ng / mL or about 25 ng / mL. In some embodiments, the incubation with the media containing the BMP-4 is for about 4 days. In some embodiments, the incubation with the media containing the bFGF / SCF / VEGF combination and the incubation with the media containing SCF / IL-3 / TPO / M-CSF / FLT3 ligand combination is for a total of about 14 days. In some embodiments, the incubation with the media containing the M-CSF / FLT3 ligand / GM-CSF combination is for at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, or at least 3 months, wherein the harvesting of floating mononuclear phagocytes may be performed every 3-5 days and fresh media containing the M-CSF / FLT3 ligand / GM-CSF combination is re-added after each harvest.

[0021] In some embodiments, the mononuclear phagocytes are harvested by collecting floating cells from the culture every 3-5 days or about 2 times per week, which can be repeated for a total of 4-70 days or longer. In some embodiments, the mononuclear phagocytes are harvested for at least 3 weeks. In some embodiments, the mononuclear phagocytes are harvested for at least 6 weeks. In some embodiments, the mononuclear phagocytes are harvested for at least 9 week. In some embodiments, the mononuclear phagocytes are harvested for at least 12 weeks.

[0022] Methods for obtaining M2 phenotype-polarized macrophages from iPSC are thus also provided. Compositions comprising a quantity of M2 phenotype-polarized macrophages derived from iPSC are thus also provided. In some embodiments, the compositions further contain a pharmaceutically acceptable excipient or carrier.4899-6723-7253 2 Page 5 of 52 065472-001005 WOPT

[0023] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.BRIEF DESCRIPTION OF THE FIGURES

[0024] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0025] Figures 1A-1H depict stepwise induction of iPSCs to anti-inflammatory iMacrophages (iMac-M2). (1A and IB) Schematic illustration and representative brightfield images of iPSC anti-inflammatory macrophages stepwise induction using chemically defined media and small molecules. From iPSC to CD14+ iMonocyte (1A) and from iMonocyte to CD163+ / CD206+ iMacrophage-M2 (IB). (1C) Number of iMonocytes harvested per day of culture in 6 wells. (ID) iMonocyte cell viability (in percentage) at repeated harvests 2 times per week. (IE) Percentage of CD14+ iMonocytes from the harvest. (IF) Representative FACS plots of each of CD14 and CD68 at different stages: iMonocytes, iMacrophage (iMac), and iMac-M2. (1G) Representative FACS plots of CD163 different stages: iMonocytes, iMacrophage (iMac), and iMac-M2. (1H) Representative fluorescent images demonstrating iMac-M2 phagocytosis activity and a dots plot showing the percentage of Zymosan phagocyted.

[0026] Figure 2A depicts that transcriptomic profiling reveals M2-specific gene expression signature in iMac-M2 cells. Figure 2B depicts flow cytometry analysis of CD14, CD68, and CD 163 expression levels from Day 7 to Day 34 post-differentiation.

[0027] Figures 3A-3E depict in vitro anti-inflammatory effects of iMac-M2 on chondrocytes and synoviocytes from osteoarthritic patients in vitro. (3A) Schematic representation of the in vitro osteoarthritix model. (3B-3C) Gene expression levels of pro-inflammatory cytokines (IL-6, IL-ip, and IL-8) 24 hours after inducing inflammation in chondrocytes (3B; “Ch”) and fibroblast-like synoviocytes (“FLS”) (3C) (labeled as “Ch-If” or “FLS-If ’, respectively), or further co-cultured with iMac-M2 cells (labeled as “Ch-If + iMac-M2” or “FLS-If + iMac-M2”, respectively), in comparison to chondrocytes or FLS without inflammation induction. (3D-3E) Gene expression levels of proteolytic enzyme (ADAMTS5, MMP3, MMP9, MMP13) 14 days post-inflammation in chondrocytes (3D) or in FLS (3E).

[0028] Figures 4A-4L depict phenotypic stability of iMac-M2 macrophages under inflammatory conditions. (4A) Schematic hypothesis: Inflamed chondrocytes exposed to IL-ip and LPS adopt an osteoarthritic-like phenotype and release high levels of pro-inflammatory4899-6723-7253 2 Page 6 of 52 065472-001005WOPTcytokines (IL-6, TNFa). We hypothesized that these inflammatory mediators may influence iPSC-derived M2 macrophages (iMac-M2), potentially driving them toward a pro-inflammatory Ml -like phenotype. (4B) Representative brightfield images of iMac-M2 morphology under control (non-inflamed) or inflammatory conditions (IL-6, TNFa, IL-ip, or LPS) at Day 3, Day 7, and Day 14. (4C-4D) Flow cytometry analysis of M2 markers CD163 (4C) and CD206 (4D) expressed by iMac-M2 at Days 3, 7, and 14 following exposure to inflammatory stimuli. (4E-4F) Flow cytometry quantification of Ml markers CD64 (4E) and CD80 (4F) under the same conditions and timepoints. (4G-4I) Gene expression of M2-associated genes (Stat3 in 4G, cMyc in 4H, and Stat6 in 41) measured by RT-qPCR across the same inflammatory conditions and timepoints. (4J-4L) Gene expression of Ml -associated genes (Statl in 4 J, HIFla in 4K, and Stat5 in 4L) showing a dynamic regulation depending on stimulus and duration. Data are presented as relative quantification (RQ) compared to control (non-inflamed) iMac-M2. Statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0 001, ****p < 0.0001.

[0029] Figures 5A-5E depict in vivo administration of iMac-M2 in immunocompetent rats following DMM surgery. (5A) Schematic representation of the experimental design. DMM was performed on the right knee of each animal. One week post-surgery, PBS or iMac-M2 were injected. Blood samples were collected before and one week after injection to measure IgM and cytokine levels. Behavioral assessments (BBT) would be conducted every two weeks post-DMM. X-ray microtomography would be performed at 6 and 12 weeks post-DMM. Finally, 3D whole-tissue imaging of nerves and macrophages would be conducted on n=3 animals per group. Bulk RNA sequencing would be performed on n=3 rats per group, and OARSI scoring would be conducted on n=7 rats per group. (5B) Systemic IgM levels (ng / mL) measured pre-inj ection and one week post-injection with PBS (control group) and iMac-M2 treatment, normalized to baseline. (5C) Gait Behavioral tests, including von Frey (VF), was performed before DMM (baseline) and one week after iMac-M2 injection. (5D) Weight Bearing. (5E) Bulk RNA sequencing of synovium and cartilage to assess gene expression.

[0030] Figures 6A-6H depict subchondral bone changes assessed by micro-CT, cartilage degeneration, and synovial inflammation evaluated by histological analysis. (6A) Longitudinal micro-CT imaging of subchondral bone structure in Sham, DMM-Saline, and DMM-iMac-M2 groups at weeks 10 and 22. 3D reconstructions and coronal cross-sections highlight bone degeneration (arrows) in the saline-treated DMM group, whereas iMac-M2 treatment maintains bone structure over time. (6B) Quantification of bone volume fraction (BV / TV) at weeks 10 and 22. (6C) Quantification of trabecular thickness (Tb.Th) at weeks 104899-6723-7253 2 Page 7 of 52 065472-001005 WOPTand 22. (6D) Bone mineral density (BMD) quantification at weeks 10 and 22. (6E) Histological assessment of cartilage integrity using Safranin O / Fast Green staining. (6F) OARSI scoring of cartilage degeneration using Safranin O Fast Green staining. (6G) Hematoxylin & Eosin (H&E) staining of synovial tissue. (6H) Quantification of vascular density in synovial tissue.DESCRIPTION OF THE INVENTION

[0031] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rded., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7thed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4thed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see D. Lane, Antibodies: A Laboratory Manual 2nded. (Cold Spring Harbor Press, Cold Spring Harbor NY, 2013); Kohler and Milstein, (1976) Eur. J. Immunol. 6: 511; Queen et al. U. S. Patent No. 5,585,089; and Riechmann et al., Nature 332: 323 (1988); U.S. Pat. No.4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al., Nature 334:544-54 (1989); Tomlinson I. and Holliger P. (2000) Methods Enzymol, 326, 461-479; Holliger P. (2005) Nat. Biotechnol. Sep;23(9):1126-36).

[0032] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0033] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.4899-6723-7253 2 Page 8 of 52 065472-001005WOPT

[0034] The term “about” or “approximately” when used in connection with a referenced numeric indication (in percentage) means the referenced numeric indication (in percentage) plus or minus up to 5% of that referenced numeric indication (in percentage), unless otherwise specifically provided for herein. In various embodiments, the term “about” when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of that referenced numeric indication, if specifically provided for in the claims. In other embodiments, “about” or “approximately” when used in connection with a referenced numeric indication of a period of time in units of at least days (e.g., day, week, or month) means the referenced numeric indication plus or minus at least one day, or at least one day and up to 10% of the indicated period of time when 10% of the indicated period of time is greater than 1 day. For example, the language “approximately 4 days covers the range of 3 days to 5 days; the language “approximately” 60 days or “approximately” 2 months covers the range of 54 days to 66 days.

[0035] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, for example, a mammal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf. In an embodiment, the subject is mammal. The mammal can be a human, nonhuman primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. In some embodiments, the subject is human. In some embodiments, the subject is a child / under 18 years old. In some embodiments, the subject is an athlete. In some embodiments, the subject is a human with a joint injury. In some embodiments, the subject does not have degenerative arthritis. In other embodiments, the subject has degenerative arthritis.

[0036] The terms “treating” or “treatment” or “to treat” refer to therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic disease or disorder. Thus, those in need of treatment include those already with the disorder. In certain embodiments, a subject is successfully “treated” for a disease or disorder if the subject shows, e.g., total, partial, permanent, or transient, alleviation or elimination of any symptom associated with the disease or disorder.

[0037] The term “therapeutically effective quantity” or “therapeutically effective amount” refers to an amount of an immunological cell or a pharmaceutical composition described herein that is sufficient and / or effective in achieving a desired therapeutic effect in4899-6723-7253 2 Page 9 of 52 065472-001005WOPTtreating a patient having a disease. In some embodiments, a therapeutically effective amount of the immune cell will avoid adverse side effects.

[0038] The term “pluripotent stem cells” or “PSCs” refer to self-replicating cells that have the ability to develop into any of endoderm, ectoderm, and mesoderm cells, as well as growth ability. Examples of the pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, embryonic stem cells derived from a cloned embryo obtained by nuclear transfer (ntES cells), germline stem cells (“GS cells”), embryonic germ cells (“EG cells”), and induced pluripotent stem cells (“iPS cells” or “iPSCs”). In some embodiments PSCs are human PSCs. Preferred examples of the PSCs include iPS cells.

[0039] Induced pluripotent stem (iPS) cells can be prepared by introducing specific reprogramming factors to somatic cells, which reprogramming factors are in the forms of DNAs or proteins. iPS cells are somatic cell-derived artificial stem cells having properties almost equivalent to those of ES cells, such as pluripotency of differentiation and growth ability by self-renewal. The reprogramming factors may be constituted by genes or gene products thereof, or non-coding RNAs, which are expressed specifically in ES cells; or genes or gene products thereof, non-coding RNAs or low molecular weight compounds, which play important roles in maintenance of the undifferentiated state of ES cells. Examples of the genes of the reprogramming factors include Oct3 / 4, Sox2, Soxl, Sox3, Soxl5, Soxl7, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbxl5, ERas, ECAT15-2, Tell, beta-catenin, Lin28b, Salll, Sall4, Esrrb, Nr5a2 and Tbx3, and these reprogramming factors may be used either alone or in combination.

[0040] The term “serum” refers to human serum, monkey serum, fetal bovine serum, bovine serum, pig serum, equine serum, donkey serum, chicken serum, quail serum, sheep serum, goat serum, dog serum, cat serum, rabbit serum, rat serum, guinea pig serum, mouse serum, and the like. Examples of the medium which does not contain serum include minimum essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), Iscove’s modification of Dulbecco’s medium (IMDM), StemPro-34SFM (Invitrogen), Stemline II (Sigma-Aldrich) and the like which are supplemented with ITS; medium for culturing primate ES cells (medium for primate ES / iPS cells, ReproCELL) wherein a serum alternative has been preliminarily added; and serum-free medium (mTeSR, Stemcell Technology). The medium which does not comprise serum, or “serum-free”, is more preferably mTeSRl medium or StemPro-34 serum-free medium.

[0041] The term “differentiation” is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell such as, for4899-6723-7253 2 Page 10 of 52 065472-001005 WOPTexample, a blood cell or a muscle cell. A differentiated or differentiation-induced cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell. The term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.

[0042] The term “differentiation marker gene,” or “differentiation gene,” refers to genes whose expression are indicative of cell differentiation occurring within a cell, such as a pluripotent cell.

[0043] The term “adhere” refers to cells attaching to a vessel, for example, a cell attaching to a sterile plastic (or coated plastic) cell culture dish or flask in the presence of an appropriate culture medium. Certain classes of cells are not sustained or do not grow in a culture unless they adhere to the cell culture vessel. Certain classes of cells (“non-adherent cells”) are maintained and / or proliferate in culture without adhering.

[0044] Culture” or “cell culture” refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. “Cell culture media,” “culture media” (singular “medium” in each case), “supplement” and “media supplement” refer to nutritive compositions that cultivate cell cultures.

[0045] Cultivate,” or “maintain,” refers to the sustaining, propagating (growing) and / or differentiating of cells outside of tissue or the body, for example in a sterile plastic (or coated plastic) cell culture dish or flask. “Cultivation,” or “maintaining,” may utilize a culture medium as a source of nutrients, hormones and / or other factors helpful to propagate and / or sustain the cells.

[0046] A “dissociated” cell refers to a cell that has been substantially separated or purified away from other cells or from a surface (e.g., a culture plate surface). For example, cells can be dissociated from an animal or tissue by mechanical or enzymatic methods.

[0047] Alternatively, cells that aggregate in vitro can be dissociated from each other, such as by dissociation into a suspension of clusters, single cells or a mixture of single cells and clusters, enzymatically or mechanically. In yet another alternative embodiment, adherent cells are dissociated from a culture plate or other surface. Dissociation thus can involve breaking cell interactions with extracellular matrix (ECM) and substrates (e.g., culture surfaces), or breaking the ECM between cells.4899-6723-7253 2 Page 11 of 52 065472-001005 WOPT

[0048] Macrophage polarization is a process by which macrophages adopt different functional programs in response to the signals from their microenvironment. Macrophage phenotypes are broadly divided into 2 groups: Ml (classically-activated macrophages) and M2 (alternatively-activated macrophages, or M2 anti-inflammatory macrophages). M2 anti-inflammatory macrophages may be activated by anti-inflammatory molecules such as IL-4, IL-13, and / or IL-10.

[0049] A “polarized macrophage” is a macrophage that correlates with an Ml or M2 macrophage phenotype. Generally Ml polarized macrophages secrete IL-12 and IL-23. The determination of a macrophage as polarized to Ml may be performed by measuring the expression of IL-12 and / or IL-23 using a standard cytokine assay and comparing that expression to the expression by newly differentiated unpolarized macrophages. Alternatively, the determination can be made by determining if the cells are CD14+, CD80+, CD206+, and CD163-. Generally M2 polarized macrophages secrete IL-10. The determination of a macrophage as polarized to M2 may be performed by measuring the expression of IL- 10 using a standard cytokine assay and comparing that expression to the expression by newly differentiated unpolarized macrophages. Alternatively, the determination can be made by determining if the cells are CD14+, CD80-, CD206+, and CD163+.

[0050] Joints are made of bones and the connective tissues that hold them together, including cartilage, tendons, ligaments, and nerves. Joints may also be classified into three categories depending on how much connective tissue they contain: fibrous joints (usually containing a lot of collagen and aren’t very flexible), cartilaginous joints (cushioned by a layer of cartilage that joins the bones together, capable of some movement), and synovial joints (having a cavity in one bone that another bone fits into, lined by a synovial membrane, and having hyaline cartilage cover ends of bones, therefore having most freedom to move). There are six types of synovial joints: hinge joints (e.g., knees and elbows), ball and socket joints (e.g., shoulders and hips), condyloid joints (e.g., wrist and the joints where toes meet the rest of foot), pivot joints (e.g., pivot joint in neck allows the head to move from one side to another), planar joints (e.g., carpal bones that join wrist to forearm and the joints between vertebrae in spine), and saddle joints (e.g., joint where thumb joints hand). A joint injury may include a damaged cartilage, damaged tendon, damaged ligament, or damaged nerve; anything that damages the bones or connective tissue. For example, arthritis, osteoarthritis, bursitis, tendinitis, and osteoporosis can affect joints, and are examples of joint injuries.

[0051] Osteoarthritis is wearing out of joint surface cartilage overtime. Post-traumatic osteoarthritis (PTOA) is wearing out of a joint that has had a physical injury. PTOA is a4899-6723-7253 2 Page 12 of 52 065472-001005 WOPTdebilitating consequence of intraarticular fractures. Diagnosis of osteoarthritis includes X-ray imaging, MRI, or CT scan, which can show loss of joint space, bone damage, bone remodeling, and bone spurs, or magnetic resonance imaging, which can show damage to soft tissues in and around the joint. Symptoms of post-traumatic arthritis can include joint pain, joint swelling, limited range of motion in joint, and / or difficulty bearing weight on joint.

[0052] The immune system plays a pivotal role in PTOA pathogenesis, with macrophages emerging as central regulators of post-injury inflammation. Synovial macrophages, which normally contribute to joint homeostasis through debris clearance and tissue repair, become aberrantly activated following injury. This activation leads to an imbalance between pro-inflammatory Ml macrophages and anti-inflammatory, pro-regenerative M2 macrophages.

[0053] The dominance of Ml macrophages sustains chronic inflammation, driving synovial fibrosis, cartilage breakdown, subchondral bone damage, and persistent pain. Although cytokine levels often decline over time, certain mediators such as IL-6 and TNF-a can remain elevated for months or years, while anti-inflammatory regulators such as IL-IRa may remain insufficient. Failure to resolve post-traumatic inflammation therefore represents a key mechanistic driver of PTOA progression.

[0054] Thus we believe that restoring macrophage polarization balance is a promising therapeutic strategy for PTOA. By enhancing M2 macrophage activity while limiting Ml-driven inflammation, it may be possible to promote tissue repair, mitigate pain, and preserve joint structure. However, current PTOA treatments remain largely surgical and symptomatic, providing limited protection against long-term disease progression.

[0055] Induced pluripotent stem cells (iPSCs) provide a scalable and clinically relevant platform for regenerative immunotherapies, allowing for the controlled generation of specialized immune cells at scale. iPSC-derived M2 macrophages (iMac-M2) represent a particularly attractive approach, as they secrete anti-inflammatory cytokines (e.g., IL-10, TGF-P), suppress catabolic mediators such as TNF-a and IL-ip, and promote tissue repair through growth factor release. We believe by directly reprogramming the post-injury immune environment, iMac-M2 therapy has the potential to intervene at an early, disease-modifying stage of PTOA.

[0056] In parallel, in vitro PTOA models using inflamed human chondrocytes and synoviocytes provide valuable platforms for mechanistic studies and therapeutic screening. These systems enable controlled investigation of inflammatory signaling, matrix degradation, and immune-stromal cell interactions relevant to PTOA pathogenesis.4899-6723-7253 2 Page 13 of 52 065472-001005 WOPT

[0057] Pain represents a defining and debilitating feature of PTOA, arising from the combined effects of inflammation, structural joint damage, and aberrant innervation. Inflammatory mediators sensitize peripheral nociceptors, while abnormal nerve sprouting, including increased calcitonin gene-related peptide (CGRP)-positive fibers, amplifies pain signaling within the joint.

[0058] The destabilization of the medial meniscus (DMM) model is a well-established preclinical model that recapitulates key features of human PTOA, including joint instability, progressive cartilage degeneration, and pain-related behaviors. Given the higher prevalence and accelerated progression of PTOA in males, the Example section includes study with male rats to reflect clinically relevant disease dynamics.

[0059] We conceive that early intra-articular administration of iPSC-derived M2 macrophages can prevent or delay PTOA onset by rebalancing post-traumatic inflammation, limiting tissue degeneration, and reducing pain. An objective of this disclosure is to use iMac-M2 therapy as a disease-modifying strategy for PTOA, including assaying its immunomodulatory, structural, and functional effects in in vitro PTOA models and in vivo DMM rat model.

[0060] We herein provide a new concept of preventive / intervening therapy using antiinflammatory macrophages. iPSCs were previously differentiated to monocytes and used for systemic injections. Here we specifically differentiate them to iMac-M2s and using as an embodiment closely after ACL or meniscus injury to prevent future osteoarthritis. In various embodiments, human iPSCs are differentiated into floating iMonocytes, harvested twice weekly, then into iMacrophages over 5 days, and finally polarized into iMac-M2 cells over 2 days.

[0061] Inflammation is known to be an important factor in osteoarthritis (OA), a degenerative cartilage disease that is often associated with pain. Macrophages play a crucial role in OA and are mediated by the local microenvironment to exhibit different phenotypes and polarization states. Therefore, polarization of iMacs at different inflammatory stages might account for various pathological processes. Although the concept of iMac-Ml (pro-inflammatory and anti-microbial) versus iMac-M2 (anti-inflammatory) polarization provides an effective system to study macrophages in vitro, the exact definition and phenotypic transition of macrophages in in vivo studies are still less defined. Especially at early stage of inflammation, the classical concept of the M1 / M2 paradigm might be misleading, when tissueresident and monocyte-derived macrophages coexisted in the local microenvironment. Emerging studies aim to control inflammation by manipulating macrophage numbers of4899-6723-7253 2 Page 14 of 52 065472-001005 WOPTphenotypes, which includes the delivery of ex vivo activated macrophages. In pre-clinical models, normalizing the aberrant M1 / M2 ratio has been indicated as a therapeutic strategy for macrophage-involved diseases. An innovative source for the standardized in vitro generation of iMac is the induced iPSC technology. Studies have shown that iPSC differentiated into macrophages under appropriate induction conditions show similar phenotypic, functional and transcriptomic characteristics as human monocyte-derived macrophages. Advantages of the iPSC technology are the generation of polarized iMac-M2 in an unlimited number without donor age restrictions.

[0062] We conceive and show that polarized iMAC-M2 therapy modify the pro-inflammatory environment of ACL-torn knees, and hinder knee OA onset, progression, and associated pain through normalization of an aberrant M1 / M2 ratio; and that the iMAC-M2 therapy can be used as a preventative treatment for patients who had an ACL or meniscus injury to prevent future osteoarthritis.

[0063] Various embodiments provide compositions comprising a quantity of macrophages polarized to an M2 phenotype (also referred to as M2 phenotype-polarized macrophages, or M2 macrophages, or anti-inflammatory macrophages) obtained from pluripotent stem cells, especially from induced pluripotent stem cells (iPSCs). In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In various aspects, the M2 phenotype-polarized macrophages obtained from the iPSCs express M2 phenotype markers for at least 7 days. In various aspects, the M2 phenotype-polarized macrophages obtained from the iPSCs express M2 phenotype markers for at least 13 days. In various aspects, the M2 phenotype-polarized macrophages obtained from the iPSCs express M2 phenotype markers for at least 16 days. In various aspects, the M2 phenotype-polarized macrophages obtained from the iPSCs express M2 phenotype markers for at least 19 days. In various aspects, the M2 phenotype-polarized macrophages obtained from the iPSCs express M2 phenotype markers for at least 34 days. In various aspects, the M2 phenotype-polarized macrophages obtained from the iPSCs co-express or are positive for CD14, CD68, and CD163 for at least 7 days. In various aspects, the M2 phenotype-polarized macrophages obtained from the iPSCs co-express or are positive for CD14, CD68, and CD163 for at least 13 days. In various aspects, the M2 phenotype-polarized macrophages obtained from the iPSCs co-express or are positive for CD14, CD68, and CD163 for at least 16 days. In various aspects, the M2 phenotype-polarized macrophages obtained from the iPSCs co-express or are positive for CD14, CD68, and CD163 for at least 19 days. In various aspects, the M2 phenotype-polarized macrophages obtained from the iPSCs co-express or are positive for CD14, CD68, and CD1634899-6723-7253 2 Page 15 of 52 065472-001005 WOPTfor at least 34 days. In various embodiments, the M2 phenotype-polarized macrophages obtained from the iPSCs retain anti-inflammatory characteristics (e.g., positive for CD14, CD68 and CD163, or reduces IL6, ILip, and / or IL8 expression levels in chondrocytes or synoviocytes) even after prolonged culture, indicating that iPSC-derived macrophages may acquire a reinforced polarization state that is less susceptible to environmental reprogramming. This behavior contrasts with primary human macrophages, which exhibited pronounced plasticity under inflammatory challenge, losing M2 markers and acquiring pro-inflammatory traits in response to TNF-a and LPS.

[0064] In various embodiments, the M2 phenotype-polarized macrophages derived from iPSCs (denoted “iMac-M2”) is effective to inhibit post-traumatic osteoarthritis. In some embodiments, the iMac-M2 inhibits inflammatory cytokine expression in inflamed chondrocytes and / or inflamed synoviocytes. In some embodiments, the iMac-M2 inhibits or reduces level(s) of IL6, ILip, and / or IL8 in inflamed chondrocytes and / or inflamed synoviocytes. In some embodiments, the iMac-M2 reduces expression levels of IL6, ILip, and IL8 in chondrocytes or synoviocytes under inflammatory stimulation, compared to the chondrocytes or synoviocytes under the inflammatory stimulation without co-culture with the iMac-M2. In some embodiments, the iMac-M2 inhibits or reduces proteolytic enzyme expression in inflamed chondrocytes and / or inflamed synoviocytes. In some embodiments, the iMac-M2 inhibits ADAMTS5, MMP3, MMP9, and / or MMP13 level(s) in the inflamed chondrocytes and / or the inflamed synoviocytes. In some embodiments, the iMac-M2 reduces expression levels of ADAMTS5, MMP3, MMP9, and MMP13 in chondrocytes or synoviocytes under inflammatory stimulation, compared to the chondrocytes or synoviocytes under the inflammatory stimulation without co-culture with the iMac-M2. Exemplary inflammatory stimulation includes physical damage or exposure to IL-6, TNFa, ILip, lipopolysaccharide, or a combination thereof. In various aspects, “inflamed” chondrocytes and / or synoviocytes, or these cells under inflammatory stimulation, may be obtained from a patient with osteoarthritis.

[0065] In various embodiments, the iMac-M2 are obtained by a process comprising: a. incubating isolated mononuclear phagocytes derived from pluripotent stem cells with a first culture media comprising macrophage colony-stimulating factor (M-CSF), optionally for about 5 days, wherein the mononuclear phagocytes derived from pluripotent stem cells differentiate into cells of macrophage lineage which express CD68;b. incubating the cells of the macrophage lineage with a second culture media comprising M-CSF, IL-4, IL-6, and IL-13, optionally for about 2 days, wherein the cells of the macrophage lineage is polarized to an M2 phenotype; and4899-6723-7253 2 Page 16 of 52 065472-001005 WOPTc. recovering (or harvesting) the cells of the macrophages lineage polarized to the M2 phenotype, wherein the cells of the macrophage lineage polarized to the M2 phenotype coexpress CD 163 and CD206, thereby obtaining the iMac-M2.

[0066] In some embodiments, the mononuclear phagocytes from which the iMac-M2 are derived are obtained by a process comprising:i. incubating induced pluripotent stem cells with a culture media comprising bone morphogenetic protein (BMP-4) to obtain cells in the form of embryoid bodies;ii. incubating the cells of the form of embryoid bodies with a first serum-free culture media comprising basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stem cell factor (SCF);iii. incubating the cells after step (ii) with a second serum-free culture media comprising SCF, IL-3, thrombopoietin (TPO), macrophage colony-stimulating factor (M-CSF), and Fms-like tyrosine kinase 3 ligand (FLT3 ligand);iv. incubating the cells after step (iii) with a third serum-free culture media comprising FLT3 ligand, GM-CSF, and M-CSF, thereby obtaining mononuclear phagocytes derived from pluripotent stem cells; andv. recovering (or harvesting) the mononuclear phagocytes after step (iv), thereby obtaining isolated mononuclear phagocytes derived from pluripotent stem cells, wherein the mononuclear phagocytes derived from pluripotent stem cells express CD14.

[0067] In some embodiments, the mononuclear phagocytes from which the iMac-M2 are derived are obtained by a process described in US20240358751, which is incorporated by reference in its entirety.

[0068] In various embodiments, iPSCs in large undifferentiated colonies are collected (e.g., by enzymatic treatment) and cultured in suspension (e.g., embryoid body (EB) generation medium) to form EBs. Large EBs are attached onto coated plates (e.g., gelatin coated plates) and cultured in a monocyte differentiation medium (typically comprising M-CSF and IL-3) until visible and viable monocytes are observed. Floating monocytes are collected, and fresh monocyte differentiation medium is replaced every 3-5 days, during which time floating monocytes are harvested. In various embodiments, the collection of floating monocytes (also called harvesting monocytes) is repeatedly performed for a time span of 1-12 weeks (e.g., see figures 1C-1E). In some embodiments, the collection of floating monocytes is repeatedly performed every 3-5 days for 1-44 weeks. In some embodiments, the collection of floating monocytes is repeatedly performed for at least 5 weeks and up to 44 weeks. In some4899-6723-7253 2 Page 17 of 52 065472-001005 WOPTembodiments, the collection of floating monocytes is repeatedly performed for at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks. Monocytes are plated in M2 differentiation medium comprising M-CSF and differentiated, then polarized into M2 phenotype using macrophage polarization medium comprising IL-4, IL-6, IL-13, and M-CSF. In various embodiments, monocytes in the sixth and subsequent harvests, wherein harvesting is performed every 3-5 days on floating monocytes, have a cell viability of at least 60% or on average 80% or more, and at least 60% of the harvested cells are CD14 positive.

[0069] In various embodiments, compared to unpolarized macrophages (iMac), the M2 phenotype-polarized macrophage lineage cells exhibit increased expression of M2-associated and anti-inflammatory genes, including CD 163, CD209, MRC1, PPARG, IL 10, CCL13, CCL18, CLEC10A, and ALOX15.

[0070] Exemplary culture mediums are described in Example 2.

[0071] For example, mTeSRl (also referred to as “mTeSR” in Example 2) is used as the culture medium supplemented with BMP4 in step (i). “TeSR” is a serum-free, xeno-free medium shown to support derivation and long-term feeder-independent culture of hPSCs, and was developed by Tenneille Ludwig and colleagues (Ludwig TE et al., Nat Biotechnol. 24: 185-7, 2006). The formulation of “TeSR” included high levels of bFGF, together with TGF, GABA, pipecolic acid, and lithium chloride. This original publication by Ludwig et al., described the use of cell support matrix composed of four human components (collagen IV, fibronectin, laminin, and vitronectin). Ludwig and colleagues further developed modifications to the medium (“mTeSRl”), which does include some animal-sourced proteins yet retains the advantages of being fully-defined and serum-free and supports the self-renewal of hPSCs without requiring feeder cells (Ludwig TE, et al., Nat Methods 3: 637-46, 2006). A mTeSRl medium, according to Ludwig TE, et al., Nat Methods 3: 637-46, 2006, contains: DMEMZF12, Stock B (including dissolved bovine serum albumin, thiamine, reduced glutathione, L-ascorbic acid 2-phosphate magnesium salt, selenium, Trace Elements B, Trace Elements C, insulin, holo-transferrin), zebrafish bFGF, TGFpi, pipecolic acid, GABA, lithium chloride, lipid, L-glutamine-P mercaptoethanol, MEM NEAA, and NaHCCE, which upon mixing is adjusted for pH to be 7.4 using NaOH and for osmolarity to be between 340 and 350 mOsMol using crystalline NaCl, and preferably filter-sterilized before use.

[0072] As another example, StemPro-34 may be used as the culture medium to be supplemented with the cytokines or reagents in steps (ii), (iii), and (iv). Alternatively, any suitable hematopoietic cell medium can be used as the mediums in at least steps (ii), (iii), and4899-6723-7253 2 Page 18 of 52 065472-001005 WOPT(iv) to be supplemented with cytokines or reagents specifically listed. The composition of StemPro-34 medium is known in the art and described in, for example, EP 0891419 (or US20040072349, US20100297090) entitled “Hematopoietic Cell Culture Nutrient Supplement” and WO1997033978 (or US20040072349, US20100297090), the contents of which are hereby incorporated by reference. However, one of skill in the art will recognize that there are several other types of media that are equivalent to StemPro-34 medium in terms of their suitability for use in culturing hematopoietic cells.

[0073] In some embodiments, the iMac-M2 are produced in a process without the addition of a GSK3 inhibitor; i.e., the culture media does not contain a GSK3 inhibitor. Exemplary GSK3 inhibitors include CHIR-99021, 6-bromoindirubin-3 '-oxime (BIO), SB216763, CHIR-98014, TWS119, IM- 12, 1-Azakenpaullone, AR-A014418, SB415286, AZD1080, AZD2858, indirubin, A 1070722, TCS 2002, Tideglusib, or any derivatives thereof.

[0074] In various embodiments, the mononuclear phagocytes derived from iPSCs (iMP) which are used to generate M2 phenotype-polarized macrophages described herein have differential gene expression compared to naturally occurring monocytes or naturally occurring macrophages or naturally occurring mononuclear phagocytes. The generated iMPs may be positive for similar markers and behave similarly in function tests especially in vivo, as the naturally occurring counterparts, thereby having at least the therapeutic efficacy as described in WO2023023346. In various aspects, neither the iPSCs nor the naturally occurring macrophages / monocytes are proliferative, but the iMP provides cysts differentiated from iPSCs, so that iMPs can bud off from the cysts and thereby be expanded in production to therapeutic quantities (or clinically meaning quantities). As a result, M2 phenotype-polarized macrophages generated from the iMP are purified and in scalable quantities.

[0075] The pharmaceutical compositions can contain a pharmaceutically acceptable excipient or carrier, such as buffers, salts, polymers, proteins, and preservatives which are added to stabilize the cells or to provide physiological osmolality. “Carrier” or “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. The carrier is suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits. “Pharmaceutically acceptable excipient” means an excipient that is useful4899-6723-7253 2 Page 19 of 52 065472-001005 WOPTin preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such carrier may be solid, liquid, semisolid. The final harvest of cells prior to formulation and patient use may also carry residual amounts of cell culture supplements. Therefore, a composition disclosed herein may include excipients, which refer to components used in the formulation and to ancillary materials (e.g., cell culture supplements) that may remain in the final product. Examples of excipients include but are not limited to human serum albumin, dimethyl sulfoxide (DMSO), calcium chloride, potassium chloride, sodium chloride, sodium lactate, water, dextran and combinations thereof.

[0076] Methods for producing macrophages polarized to an M2 phenotype from iPSCs are also provided. In various embodiments, these methods include the procedures depicted in figures 1A, IB, Example 1, and / or Example 2.

[0077] In some embodiments, a method for obtaining M2 phenotype-polarized macrophages derived from iPSCs includes:a. incubating isolated mononuclear phagocytes derived from pluripotent stem cells with a first culture media comprising macrophage colony-stimulating factor (M-CSF), optionally for about 5 days, wherein the mononuclear phagocytes derived from pluripotent stem cells differentiate into cells of macrophage lineage which express CD68;b. incubating the cells of the macrophage lineage with a second culture media comprising M-CSF, IL-4, IL-6, and IL-13, optionally for about 2 days, wherein the cells of the macrophage lineage is polarized to an M2 phenotype; andc. recovering the cells of the macrophages lineage polarized to the M2 phenotype, wherein the cells after step (c) express CD 163 and / or CD206.

[0078] In some embodiments, the isolated mononuclear phagocytes derived from pluripotent stem cells are obtained by:i. incubating induced pluripotent stem cells with a third culture media comprising bone morphogenetic protein (BMP-4) to obtain cells in the form of embryoid bodies;ii. incubating the cells of the form of embryoid bodies with a first serum-free culture media comprising basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stem cell factor (SCF);iii. incubating the cells after step (ii) with a second serum-free culture media comprising SCF, IL-3, thrombopoietin (TPO), macrophage colony-stimulating factor (M-CSF), and Fms-like tyrosine kinase 3 ligand (FLT3 ligand);4899-6723-7253 2 Page 20 of 52 065472-001005 WOPTiv. incubating the cells after step (iii) with a third serum-free culture media comprising M-CSF, GM-CSF, and FLT3 ligand, thereby obtaining mononuclear phagocytes derived from pluripotent stem cells; andv. recovering the mononuclear phagocytes, thereby obtaining isolated mononuclear phagocytes derived from pluripotent stem cells.

[0079] In additional embodiments, the recovering of the step (v) comprises collecting floating cells from step (iv). In some embodiments, the recovering of the step (v) comprises collecting floating cells every 3-5 days from the incubation of step (iv). In some embodiments, the recovering of the step (v) comprises collecting floating cells every 3-5 days from the incubation of step (iv) repeatedly for 2-30 times or for a time span from 4-70 days. In some embodiments, the recovering of the step (v) comprises collecting floating cells every 3-5 days from the incubation of step (iv) for at least 5 weeks and up to 44 weeks.

[0080] In some embodiments, the step (i) comprises the BMP4 at about 60-100 ng / mL; the step (ii) comprises the bFGF at about 20-30 ng / mL, the SCF at about 80-120 ng / mL, and the VEGF at about 60-100 ng / mL; the step (iii) comprises the SCF at about 40-60 ng / mL, the IL-3 at about 10-60 ng / mL, the TPO at about 1-10 ng / mL, the M-CSF at about 40-60 ng / mL, and the FLT3 ligand at about 40-60 ng / mL; the step (iv) comprises the M-CSF at about 40-60 ng / mL, the FLT3 ligand at about 40-60 ng / mL, the GM-CSF at about 20-30 ng / mL.

[0081] In some embodiments, the step (i) comprises the BMP4 at about 60-70 ng / mL. In some embodiments, the step (i) comprises the BMP4 at about 70-80 ng / mL. In some embodiments, the step (i) comprises the BMP4 at about 80-90 ng / ml. In some embodiments, the step (i) comprises the BMP4 at about 90-100 ng / mL. In some embodiments, the SCF in step (ii) is at about 80-90 ng / mL. In some embodiments, the SCF in step (ii) is at about 90-100 ng / mL. In some embodiments, the SCF in step (ii) is at about 100-110 ng / mL. In some embodiments, the SCF in step (ii) is at about 110-120 ng / mL. In some embodiments, the VEGF in step (ii) is about 60-70 ng / mL. In some embodiments, the VEGF in step (ii) is about 70-80 ng / mL. In some embodiments, the VEGF in step (ii) is about 80-90 ng / mL. In some embodiments, the VEGF in step (ii) is about 90-100 ng / mL. In some embodiments, the SCF in step (iii) is at about 40-50 ng / mL. In some embodiments, the SCF in step (iii) is at about 50-60 ng / mL. In some embodiments, the IL-3 in step (iii) is at about 15-25 ng / mL. In some embodiments, the IL-3 in step (iii) is at about 10-20 ng / mL. In some embodiments, the IL-3 in step (iii) is at about 20-30 ng / mL. In some embodiments, the IL-3 in step (iii) is at about 30-40 ng / mL. In some embodiments, the IL-3 in step (iii) is at about 40-50 ng / mL. In some embodiments, the IL-3 in step (iii) is at about 50-60 ng / mL. In some embodiments, the TPO in4899-6723-7253 2 Page 21 of 52 065472-001005 WOPTstep (iii) is at about 1-5 ng / mL. In some embodiments, the TPO in step (iii) is at about 5-10 ng / mL. In some embodiments, the M-CSF in step (iii) is at about 40-50 ng / mL. In some embodiments, the M-CSF in step (iii) is at about 50-60 ng / mL. In some embodiments, the FLT3 ligand in step (iii) is at about 40-50 ng / mL. In some embodiments, the FLT3 ligand in step (iii) is at about 50-60 ng / mL. In some embodiments, the M-CSF in step (iv) is at about 40-50 ng / mL. In some embodiments, the M-CSF in step (iv) is at about 50-60 ng / mL. In some embodiments, the FLT3 ligand in step (iv) is at about 40-50 ng / mL. In some embodiments, the FLT3 ligand in step (iv) is at about 50-60 ng / mL. In some embodiments, the GM-CSF is step (iv) at about 20-30 ng / mL.

[0082] In some embodiments, the step (i) comprises the BMP4 at about 80 ng / mL; the step (ii) comprises the bFGF at about 25 ng / mL, the SCF at about 100 ng / mL, and the VEGF at about 80 ng / mL; the step (iii) comprises the SCF at about 50 ng / mL, the IL-3 at about 20 ng / mL, the TPO at about 5 ng / mL, the M-CSF at about 50 ng / mL, and the FLT3 ligand at about 50 ng / mL; the step (iv) comprises the M-CSF at about 50 ng / mL, the FLT3 ligand at about 50 ng / mL, the GM-CSF at about 25 ng / mL.

[0083] In some embodiments, the step (a) includes the incubating with the first culture media comprising the M-CSF at 5 to 50 ng / mL; and the step (b) includes the incubating with the second culture media comprising the M-CSF, the IL-4, the IL-6, and the IL-13, each at 5 to 50 ng / mL.

[0084] In some embodiments, the M-CSF in step (a) is about 5-10 ng / mL. In some embodiments, the M-CSF in step (a) is about 10-20 ng / mL. In some embodiments, the M-CSF in step (a) is about 20-30 ng / mL. In some embodiments, the M-CSF in step (a) is about 30-40 ng / mL. In some embodiments, the M-CSF in step (a) is about 40-50 ng / mL. In some embodiments, the M-CSF, the IL-4, the IL-6, and the IL-13 in step (b) are each 5-10 ng / mL. In some embodiments, the M-CSF, the IL-4, the IL-6, and the IL- 13 in step (b) are each 10-20 ng / mL. In some embodiments, the M-CSF, the IL-4, the IL-6, and the IL- 13 in step (b) are each 20-30 ng / mL. In some embodiments, the M-CSF, the IL-4, the IL-6, and the IL- 13 in step (b) are each 30-40 ng / mL. In some embodiments, the M-CSF, the IL-4, the IL-6, and the IL-13 in step (b) are each 40-50 ng / mL.

[0085] In some embodiments, the step (a) includes the incubating with the first culture media comprising the M-CSF at about 20 ng / mL; and the step (b) includes the incubating with the second culture media comprising the M-CSF, the IL-4, the IL-6, and the IL-13, each at about 20 ng / mL.4899-6723-7253 2 Page 22 of 52 065472-001005 WOPT

[0086] In some embodiments, the step (a) includes the incubating for about 5 days with the first culture media comprising the M-CSF at about 20 ng / mL; and the step (b) includes the incubating for about 2 days with the second culture media comprising the M-CSF, the IL-4, the IL-6, and the IL-13, each at about 20 ng / mL.

[0087] In various aspects, the M2 phenotype-polarized macrophages is differentiated from an iPSC prepared by reprogramming blood cells, preferably peripheral blood mononuclear cells (PBMCs), from a subject. In some aspects, the iPSC is prepared by reprogramming blood cells, e.g., PBMCs, from a healthy human subject, a young human (e.g., a human at an age within the age group of 5-11, 12-16, 17-18, 19-21, 22-34, or 35-49 years old), or a young healthy human subject. In further embodiments, the M2 phenotype-polarized macrophages is differentiated from an iPSC prepared by reprogramming fibroblasts obtained from the subject. In some embodiments, the iPSC is prepared by reprogramming fibroblasts obtained from a subject to whom the resulting M2 phenotype-polarized macrophages are intended to be administered.

[0088] In some embodiments, methods for reprogramming blood cells to iPSCs are disclosed in WO2017219000, US Patent No. 10,221 ,395, and US Patent No. 10,745,671 , which are incorporated by reference herein.

[0089] In various embodiments, a clinically meaningful number of macrophages polarized to an M2 phenotype are generated from the pluripotent stem cells (e.g., induced pluripotent stem cells) in a process disclosed herein, especially via culturing in a bioreactor. A clinically meaningful amount of the M2 phenotype-polarized macrophages generated from the pluripotent stem cells, especially from iPSCs, can be stored (e.g., frozen) or maintained in culturing, for use in patient administration in a significant amount, as opposed to having to isolate M2 phenotyped macrophages from the patient and use them. Starting from pluripotent stem cells, specifically induced pluripotent stem cells, the generated M2 phenotype-polarized macrophages described herein may be less prone to genetic mutations, and may have fewer disease mutations, compared to autologous macrophages of M2 phenotype obtained from a patient or subject in need of the treatment.

[0090] Methods are also provided for reducing chondrocyte and / or synoviocyte inflammation after injury in a mammal, which includes locally administering an effective amount of the composition comprising iMac-M2 and a carrier to an injured joint of a mammal.

[0091] Methods are also provided for treating a mammal with joint injury and / or inhibiting post-traumatic osteoarthritis in the mammal, which include locally administering an4899-6723-7253 2 Page 23 of 52 065472-001005 WOPTeffective amount of the iMac-M2 composition to a mammal at risk of post-traumatic osteoarthritis as a result of injury to cartilage.

[0092] In various embodiments of these methods, the composition is preferably an injectable composition. In various embodiments, the composition preferably does not lead to an increase in blood IgM level or pain in the injured joint, hence not rejected by the joint. In various embodiments, the blood IgM level is not increased by more than 1%, 2%, 5%, or 10% after the iMac-M2 administration compared to the level before the iMac-M2 administration. In some embodiments, the blood IgM level at 7 days after the iMac-M2 administration is not increased or no more than 1%, 2%, 5%, 10%, or 20% higher compared to the level before the iMac-M2 administration. The injection may be performed within 14 days of the injury. The injection may be performed within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of the injury. In some embodiments, the administration is performed within 1 week from a joint injury. The injection is preferably performed before diagnosis of osteoarthritis to prevent, slow down, or inhibit likelihood or severity of developing osteoarthritis. In some embodiments, the joint is a knee joint. In some embodiments, the joint is a hip joint. In some embodiments, the joint is an ankle joint. In some embodiments, the joint is an elbow. In some embodiments, the joint injury is an anterior cruciate ligament (ACL) tear. In some embodiments, the joint injury is a posterior cruciate ligament (PCL) injury. In some embodiments, the joint injury is a medial collateral ligament (MCL) injury. In some embodiments, the joint injury is a lateral collateral ligament (LCL) injury. In some embodiments, the joint injury includes meniscus injury. In some embodiments, the administration is an intra-articular injection.

[0093] In various embodiments of the methods, the composition comprising a population of the iMac-M2 are administered in two or more exposures to the subject. In some implementations, the composition is administered on a weekly, biweekly, bimonthly, or monthly basis, or as needed by the subject. In some embodiments, the composition comprising a population of the iMac-M2 are administered in one dose. In some implementations, the composition in each exposure to the subject can include at least 106cells, 107cells, 108cells, 109cells, 1010cells, or 1011cells. In various implementations, the therapeutically effective dose of cells depends on a patient’s needs, age, physiological condition and healthy state, and the therapeutic goal. In some implementations, a low dose of cells is repeatedly transplanted.

[0094] Various embodiments provide methods for treating joint injury, reducing inflammation in the joint, inhibiting osteoarthritis, or reducing severity or likelihood of post-traumatic osteoarthritis are provided, which include administering to a subject in need thereof a therapeutically effective quantity of M2 phenotype-polarized macrophages derived from4899-6723-7253 2 Page 24 of 52 065472-001005 WOPTpluripotent stem cells. Methods are also provided for administering pluripotent stem cell-derived M2 phenotype-polarized macrophages in an individual in need thereof. For example, a method is provided for administering iPSC-derived M2 phenotype-polarized macrophages (iMac-M2) in an individual in need thereof, the method comprising administering iMac-M2 locally to an individual who has undergone or is undergoing a joint injury, who is experiencing joint pain or swelling, who is at risk of developing post-traumatic osteoarthritis, who has a fracture, a ligament tear, or a meniscal damage, or whose joint has elevated pro-inflammatory mediators and / or elevated cartilage degradation markers compared to a reference subject with no joint damage, pain, or osteoarthritis. In another example, a method is provided for administering iPSC-derived M2 phenotype-polarized macrophages (iMac-M2) in an individual in need thereof, the method comprising administering iMac-M2 prepared by a process described in Example 1 or 2 to the individual in need thereof, preferably within 10 days, 7 days, 3 days, 2 days, or 1 day of a joint injury that the individual has experienced or is experiencing. Preferably, the M2 phenotype-polarized macrophages are derived from iPSCs, and remain positive for CD14, CD68, and CD163 for a sustained period of time, e.g., at least 7, 13, 16, 19, or 34 days. Preferably, the iPSC-derived, M2 phenotype-polarized macrophages remain the M2 phenotype (e.g., positive for CD68 and CD 163) under sustained inflammatory stimulation / stress, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, of longer.

[0095] In some embodiments, a method for treating joint injury in an individual in need thereof is provided, which includes administering to the individual a therapeutically effective quantity of M2 phenotype-polarized macrophages derived from pluripotent stem cells.

[0096] In some embodiments, a method for reducing inflammation in a joint, or reducing synovial inflammation, in a subject includes administering to the subject a therapeutically effective quantity of M2 phenotype-polarized macrophages derived from pluripotent stem cells.

[0097] In some embodiments, a method for reducing severity or likelihood of post-traumatic osteoarthritis in a subject includes administering to the subject a therapeutically effective quantity of M2 phenotype-polarized macrophages derived from pluripotent stem cells.

[0098] In some embodiments, the methods for treating joint injury, reducing inflammation in the joint, inhibiting osteoarthritis, or reducing severity or likelihood of post-traumatic osteoarthritis reduce IL6, ILip, and / or IL8 levels in the joint or in inflamed chondrocytes and / or inflamed synoviocytes. In some embodiments, the methods for treating4899-6723-7253 2 Page 25 of 52 065472-001005 WOPTjoint injury, reducing inflammation in the joint, inhibiting osteoarthritis, or reducing severity or likelihood of post-traumatic osteoarthritis reduce nerve growth factor receptor (NGFR), nerve growth factor (NGF), and IL6 expression levels in the individual receiving the administration of the M2 phenotype-polarized macrophage lineage cells obtained from iPSCs. In some embodiments, the methods for treating joint injury, reducing inflammation in the joint, inhibiting osteoarthritis, or reducing severity or likelihood of post-traumatic osteoarthritis reduce IL6, ILip, and IL8 levels in the joint or in inflamed chondrocytes and / or inflamed synoviocytes, compared to without the administration of the iPSC-derived, M2 phenotype-polarized macrophages.

[0099] In some embodiments, the methods for treating joint injury, reducing inflammation in the joint, inhibiting osteoarthritis, or reducing severity or likelihood of post-traumatic osteoarthritis reduce ADAMTS5, MMP3, MMP9, and / or MMP13 levels in the joint or in inflamed chondrocytes and / or inflamed synoviocytes. In some embodiments, the methods for treating] oint injury, reducing inflammation in the joint, inhibiting osteoarthritis, or reducing severity or likelihood of post-traumatic osteoarthritis reduce ADAMTS5, MMP3, MMP9, and MMP13 levels in the joint or in inflamed chondrocytes and / or inflamed synoviocytes, compared to without the administration of the iPSC-derived, M2 phenotype-polarized macrophages.

[0100] In some embodiments, an individual in need of a treatment is one who has undergone or is undergoing a joint injury. In some embodiments, an individual in need of a treatment is one who is experiencing joint pain or swelling. In some embodiments, an individual in need of a treatment is one who is at risk of developing post-traumatic osteoarthritis. In some embodiments, an individual in need of a treatment is one who has a fracture, a ligament tear, or a meniscal damage. In some embodiments, an individual in need of a treatment is one whose joint has elevated pro-inflammatory mediators (e.g., IL-ip, TNF-a, IL-6, or IL- 17) and cartilage degradation markers (e.g., matrix metalloproteinases, prostaglandins, neuropeptides), compared to a reference subject with no joint damage, pain, or osteoarthritis. In some embodiments, an individual in need of a treatment does not have degenerative osteoarthritis. In some embodiments, an individual in need of a treatment is a human. In some embodiments, an individual in need of a treatment is a mammal.

[0101] In some embodiments, the methods for treating joint injury, reducing inflammation in the joint, inhibiting osteoarthritis, or reducing severity or likelihood of post-traumatic osteoarthritis include administering M2 phenotype-polarized macrophages obtained by a process comprising:4899-6723-7253 2 Page 26 of 52 065472-001005 WOPTa. incubating isolated mononuclear phagocytes derived from pluripotent stem cells with a first culture media comprising macrophage colony-stimulating factor (M- CSF), wherein the mononuclear phagocytes derived from pluripotent stem cells differentiate into cells of macrophage lineage which express CD68;b. incubating the cells of the macrophage lineage with a second culture media comprising M-CSF, IL-4, IL-6, and IL-13, wherein the cells of the macrophage lineage is polarized to an M2 phenotype; andc. recovering the cells of the macrophages lineage polarized to the M2 phenotype, wherein the cells polarized to the M2 phenotype co-express CD 163 and CD206.

[0102] In some embodiments, the methods for treating joint injury, reducing inflammation in the joint, inhibiting osteoarthritis, or reducing severity or likelihood of post-traumatic osteoarthritis include administering M2 phenotype-polarized macrophages obtained by a process comprising:i. incubating induced pluripotent stem cells with a third culture media comprising bone morphogenetic protein (BMP-4) to obtain cells in the form of embryoid bodies;ii. incubating the cells of the form of embryoid bodies with a first serum-free culture media comprising basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stem cell factor (SCF) for two days;iii. incubating the cells after step (ii) with a second serum-free culture media comprising SCF, IL-3, thrombopoietin (TPO), macrophage colony-stimulating factor (M-CSF), and Fms-like tyrosine kinase 3 ligand (FLT3 ligand) for 6-8 days;iv. incubating the cells after step (iii) with a third serum-free culture media comprising FLT3 ligand, GM-CSF, and M-CSF, thereby obtaining mononuclear phagocytes derived from pluripotent stem cells; andv. recovering the mononuclear phagocytes after step (iv), thereby obtaining isolated mononuclear phagocytes derived from pluripotent stem cells,wherein the mononuclear phagocytes derived from pluripotent stem cells express CD 14; anda. incubating isolated mononuclear phagocytes derived from pluripotent stem cells with a first culture media comprising macrophage colony-stimulating factor (M-CSF), wherein the mononuclear phagocytes derived from pluripotent stem cells differentiate into cells of macrophage lineage which express CD68;4899-6723-7253 2 Page 27 of 52 065472-001005 WOPTb. incubating the cells of the macrophage lineage with a second culture media comprising M-CSF, IL-4, IL-6, and IL-13, wherein the cells of the macrophage lineage is polarized to an M2 phenotype; andc. recovering the cells of the macrophages lineage polarized to the M2 phenotype, wherein the cells polarized to the M2 phenotype co-express CD 163 and CD206

[0103] In some embodiments, the methods for treating joint injury, reducing inflammation in the joint, inhibiting osteoarthritis, or reducing severity or likelihood of post-traumatic osteoarthritis in an individual in need thereof, include:generating M2 phenotype-polarized macrophages from iPSCs in a process described herein, andadministering to the individual a therapeutically effective amount of the M2 phenotype-polarized macrophages.

[0104] Some embodiments provide the methods for treating joint injury, reducing inflammation in the joint, inhibiting osteoarthritis, or reducing severity or likelihood of post-traumatic osteoarthritis in an individual in need thereof further include administering a cartilage-protective or regenerative therapy to the individual in need thereof. In some embodiments, the methods for treating joint injury, reducing inflammation in the joint, inhibiting osteoarthritis, or reducing severity or likelihood of post-traumatic osteoarthritis include administering iPSC-derived, M2 phenotype-polarized macrophage lineage cells to an individual who has received a cartilage-protective or regenerative therapy. Exemplary cartilage-protective or regenerative therapies include but are not limited to physical exercise such as swimming, cycling, walking, or balance training; weight management; wearing orthotic devices, braces, or footwear to redistribute forces across joint; receiving nutritional supplements such as glucosamine and chondroitin sulfate, omega-3 fatty acids, curcumin, vitamins; injecting corticosteroids, hyaluronic acid, or platelet-rich plasma; receiving nonsteroidal anti-inflammatory drugs; microfracture to stimulate new cartilage formation; osteotomy; osteochondral grafting; and autologous chondrocyte implantation; or implanting biomaterial scaffolds alone or combined with cells and growth factors.EXAMPLESThe following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.4899-6723-7253 2 Page 28 of 52 065472-001005 WOPTExample 1. Modulating Inflammation in Post-Traumatic Osteoarthritis using iPSC-derived Anti-inflammatory Macrophages.1.1 Differentiation and characterization of anti-inflammatory macrophages (iMac-M2).

[0105] Human induced pluripotent stem cells (iPSCs) were successfully differentiated into anti-inflammatory M2 macrophages (iMac-M2) through a stepwise protocol using chemically defined media and small molecules (Fig. 1A-1B). The differentiation process involved sequential stages including iMyeloid progenitors, CD14+iMonocytes, CD68+macrophages, and final polarization into M2 macrophages characterized by CD163+ / CD206+expression.

[0106] The differentiation protocol was reproducible across multiple independent iPSC lines, yielding consistent cellular populations at each stage of differentiation.

[0107] Daily harvests of iMonocytes showed stable production with high viability (>90%) over time (Fig. 1C-1D). The proportion of CD14+cells progressively increased across successive harvests (Fig. IE), indicating sustained and efficient generation of iPSC-derived monocytes.

[0108] Following polarization with IL-4, IL-6 and IL-13 IL-4, the resulting iMac-M2 population exhibited a marked increase in the expression of M2-associated markers, with 79% of cells expressing CD 163 (Fig. IF). Functional characterization demonstrated that iMac-M2 displayed robust phagocytic activity (Fig. 1G), with more than 90% of cells internalizing Zymosan particles (Fig. 1H).1.2. Distinct transcriptional profiles of iMac-M2 from iMac.

[0109] Gene expression analysis of iMac and iMac-M2 macrophages revealed distinct transcriptional profiles associated with inflammatory and tissue-regulatory states. The heatmap analysis (Fig. 2 A) showed that iMac-M2 macrophages, compared to unpolarized macrophages (iMac), exhibited increased expression of M2-associated and anti-inflammatory genes, including CD163, CD209, MRC1, PPARG, IL10, CCL13, CCL18, CLEC10A, and ALOX15.

[0110] Conversely, iMac samples showed higher expression of genes associated with inflammatory activation, including MMP9, CSF1R, and CCR2.[oni] Genes associated with macrophage motility and tissue surveillance, including CX3CR1 and FCMR, showed comparable expression levels between iMac and iMac-M2 samples, indicating that polarization was not associated with major transcriptional changes in mobility-related pathways.

[0112] In addition, genes involved in tissue remodeling and extracellular matrix regulation, such as MMP10 and MMP12, were differentially expressed between iMac and4899-6723-7253 2 Page 29 of 52 065472-001005 WOPTiMac-M2 macrophages, consistent with distinct transcriptional programs associated with inflammatory versus tissue-regulatory macrophage states.1.3. Stable iMac-M2 phenotype in culture.

[0113] To assess the temporal stability of iMac-M2 in culture, flow cytometry analysis was performed at multiple timepoints post-differentiation, from Day 7 to Day 34 (Fig. 2B).

[0114] The expression levels of CD14, CD68, and CD163 were maintained across all analyzed time points, indicating persistence of the macrophage and M2-associated phenotype during prolonged culture (Fig. 2B).

[0115] Across the culture period, iMac-M2 consistently displayed high levels of CD68 and CD163, with no marked loss of marker expression observed up to Day 34 (Fig. 2B). 1.4. iMac-M2 reduce inflammation and matrix degradation.

[0116] To evaluate the effects of iMac-M2 on joint-resident cells in vitro, primary human chondrocytes and fibroblast-like synoviocytes (FLS) isolated from osteoarthritic patients were used (Fig. 3A). Chondrocytes and FLS were exposed to IL-ip and LPS for 24 hours to induce an inflammatory state, followed by direct co-culture with iMac-M2 (Fig. 3 A). Acute inflammatory responses were assessed by measuring gene expression of pro-inflammatory cytokines 24 hours after iMac-M2 treatment.

[0117] To ensure cell type specific gene expression analysis, iMac-M2 were removed by flow cytometric sorting prior to RNA extraction, allowing for selective analysis of chondrocytes and FLS.

[0118] The results showed that iMac-M2 significantly reduced the expression of IL6, IL1B, and IL8 in inflamed chondrocytes (Fig. 3B) and FLS (Fig. 3C).

[0119] To assess longer-term effects, expression of matrix-degrading enzymes was analyzed after prolonged co-culture. iMac-M2 treatment was associated with reduced expression of ADAMTS5, MMP3, MMP9, and MMP13 in chondrocytes (Fig. 3D) and FLS (Fig. 3E).

[0120] Collectively, these data show that iMac-M2 treatment attenuates inflammatory cytokine expression and reduces matrix-degrading enzyme expression in human chondrocytes and FLS in vitro.1.5. iMac-M2 maintain phenotype under inflammatory conditions.

[0121] To assess the phenotypic stability of iMac-M2 under inflammatory conditions, cells were exposed to IL-6, TNF-a, IL-ip, or LPS, which are inflammatory mediators associated with osteoarthritic environments (Fig. 4A).4899-6723-7253 2 Page 30 of 52 065472-001005 WOPT

[0122] Phase contrast imaging showed that iMac-M2 maintained a largely stable morphology over time under control and inflammatory conditions, with only modest morphological changes observed depending on the stimulus and duration of exposure (Fig.4B).

[0123] Flow cytometry analysis showed that iMac-M2 retained expression of the M2-associated surface markers CD 163 and CD206 at Days 3, 7, and 14 across all inflammatory conditions, although stimulus-dependent variations were observed (Fig. 4C-4D).

[0124] A partial reduction in CD 163 and CD206 expression was detected following prolonged exposure to TNF-a or LPS, but M2 marker expression remained detectable at all time points analyzed (Fig. 4C-4D).

[0125] In parallel, expression of the Ml -associated surface markers CD64 and CD80 remained low overall, with transient increases observed depending on the inflammatory stimulus and time point (Fig. 4E-4F).

[0126] Gene expression analysis by RT-qPCR further characterized the transcriptional response of iMac-M2 under inflammatory conditions. Expression of M2-associated genes Stat3, cMyc, and Stat6 showed dynamic regulation across stimuli and time points, with sustained expression detected under all inflammatory conditions (Fig. 4G-4I).

[0127] Ml -associated genes Statl, HIFla, and Stat5 exhibited stimulus- and durationdependent regulation, with increased expression observed under specific inflammatory conditions (Fig. 4J-4L).

[0128] Despite transcriptional modulation of Ml-associated genes, flow cytometry analyses did not reveal a complete transition toward an Ml -dominant surface marker profile.1.6. Joint response to iMac-M2 treatment.

[0129] Immunocompetent rats underwent destabilization of the medial meniscus (DMM) surgery to induce PTOA, followed by intra-articular administration of iMac-M2 or PBS control one week post-surgery (Fig. 5 A). Blood samples were collected before injection and one week after treatment to assess systemic IgM levels.

[0130] Systemic IgM concentrations remained comparable between PBS- and iMac-M2 -treated animals before and after injection (Fig. 5B), indicating that iMac-M2 administration did not induce detectable changes in circulating IgM levels during the early post-injection period.

[0131] Functional outcomes were assessed longitudinally using gait analysis and weight-bearing measurements. PBS-treated animals exhibited a progressive increase in gait asymmetry between the operated and contralateral limbs over time (Fig. 5C). In contrast,4899-6723-7253 2 Page 31 of 52 065472-001005 WOPTanimals receiving iMac-M2 displayed reduced gait asymmetry and values closer to baseline levels throughout the follow-up period. Consistent with these observations, weight-bearing analysis showed that iMac-M2-treated rats maintained a more balanced load distribution between hind limbs compared to PBS-treated controls (Fig. 5D).

[0132] To evaluate molecular changes associated with treatment, bulk RNA sequencing was performed on synovium and cartilage harvested from PBS- and iMac-M2-treated animals (Fig. 5E). In synovial tissue, expression levels of pain-related genes including TRPV1, TNF, TAC1, and SCN9A were similar between groups, whereas NGFR, NGF, and IL6 showed lower expression in iMac-M2-treated animals compared to PBS controls. Genes associated with macrophage-related pathways, such as SOCS3 and MRC1, exhibited comparable expression levels between treatment groups.

[0133] In cartilage, expression of extracellular matrix-related genes, including MMP3, MMP9, and MMP13, did not differ significantly between PBS- and iMac-M2-treated animals (Fig. 5E), indicating limited transcriptional changes in cartilage at the analyzed time point. 1.7. iMac-M2 preserve joint structure and limit degeneration.

[0134] To determine the structural and histological impact of iMac-M2 therapy in PTOA, we evaluated subchondral bone remodeling and cartilage integrity longitudinally. Longitudinal micro-computed tomography was performed to assess subchondral bone remodeling following DMM surgery in Sham, DMM-Saline, and DMM-iMac-M2 groups at weeks 10 and 22 post-DMM (Fig. 6A). Three-dimensional reconstructions and coronal crosssections revealed marked subchondral bone degeneration in saline-treated DMM joints, characterized by trabecular disorganization and bone loss, which progressed between weeks 10 and 22. In contrast, joints treated with iMac-M2 exhibited preservation of subchondral bone architecture overtime, with structural features more closely resembling those observed in Sham controls (Fig. 6A).

[0135] Quantitative micro-CT analysis supported these observations. Bone volume fraction (BV / TV) was significantly reduced in saline-treated DMM joints compared to Sham at both time points, whereas iMac-M2 -treated joints showed higher BV / TV values relative to saline-treated animals at weeks 10 and 22 (Fig. 6B). Similarly, trabecular thickness (Tb.Th) was markedly decreased in the saline group, while iMac-M2 treatment was associated with partial preservation of trabecular thickness at both time points (Fig. 6C). Bone mineral density (BMD) followed a comparable pattern, with reduced values in saline-treated DMM joints and higher BMD measurements in iMac-M2 -treated joints, particularly at week 10 (Fig. 6D).4899-6723-7253 2 Page 32 of 52 065472-001005 WOPT

[0136] Cartilage integrity was subsequently evaluated using Safranin O / Fast Green staining (Fig. 6E). Saline-treated DMM joints exhibited clear signs of cartilage degeneration, including surface irregularities, reduced proteoglycan staining, and structural disorganization. In contrast, cartilage from iMac-M2 -treated joints showed improved preservation of tissue architecture and Safranin O staining intensity compared to saline-treated controls. These histological findings were supported by OARSI scoring, which revealed lower cartilage degeneration scores in the iMac-M2 group relative to saline-treated animals (Fig. 6F).

[0137] Synovial tissue remodeling was assessed by hematoxylin and eosin staining (Fig. 6G). Saline-treated DMM joints displayed pronounced synovial thickening and hyperplasia, consistent with ongoing synovial inflammation. In comparison, synovial tissue from iMac-M2 -treated joints appeared less expanded and exhibited a more organized morphology. Quantification of vascular density demonstrated a significant increase in synovial neovascularization in saline-treated DMM joints compared to Sham, whereas iMac-M2 treatment was associated with reduced vascular density, approaching levels observed in Sham joints (Fig. 6H).

[0138] Overall, we showed that iPSC-derived anti-inflammatory macrophages (iMac-M2) can modulate key inflammatory and structural features associated with post-traumatic osteoarthritis (PTOA). Through a combination of in vitro and in vivo experiments, we demonstrated that iMac-M2 retain a stable M2 phenotype under inflammatory stress, suppress the production of pro-inflammatory cytokines and matrix-degrading enzymes in patient-derived chondrocytes and synoviocytes, and preserve joint structure and function in a rat model of PTOA induced by destabilization of the medial meniscus (DMM).

[0139] Notably, iMac-M2 treatment was associated with behavioral improvement in weight-bearing and gait symmetry, without inducing systemic immunogenicity, supporting the translational potential of this immunomodulatory approach in an immunocompetent setting.

[0140] The stepwise differentiation protocol yielded iMac-M2 with high purity, as evidenced by robust expression of CD 163 and CD206, key markers of the M2 phenotype. However, while most existing studies evaluate macrophage polarization over a limited number of differentiation cycles or short culture windows, our study extends this analysis across 24 independent harvests (corresponding to approximately 12 weeks of sustained production), providing a more stringent assessment of long-term phenotypic stability and reproducibility. Notably, selected cultures were maintained for up to 44 weeks, during which a gradual decline in iMonocyte output was observed, while macrophage differentiation and M2 polarization were preserved.4899-6723-7253 2 Page 33 of 52 065472-001005 WOPT

[0141] The consistency of this process across multiple iPSC lines highlights the robustness of the platform, an important requirement for therapeutic development. Importantly, iMac-M2 retained their anti-inflammatory characteristics even after prolonged culture, indicating that iPSC-derived macrophages may acquire a reinforced polarization state that is less susceptible to environmental reprogramming. This behavior contrasts with primary human macrophages, which exhibited pronounced plasticity under inflammatory challenge, losing M2 markers and acquiring pro-inflammatory traits in response to TNF-a and LPS.

[0142] Sustained activation of STAT3- and STAT6-associated transcriptional programs may contribute to this phenotypic stability. This enhanced stability is particularly relevant in the context of chronic joint inflammation, where macrophages are continuously exposed to pro-inflammatory cues.

[0143] In contrast to other studies focusing on macrophage generation, short-term phenotypic characterization, or disease models outside the musculoskeletal system, our work demonstrated that iPSC-derived M2 macrophages remain phenotypically stable under sustained inflammatory stress and exert functional and structural benefits in a clinically relevant PTOA model.

[0144] In co-culture experiments, iMac-M2 effectively suppressed inflammatory cytokine production (IL-6, IL-ip, IL-8) in chondrocytes and synoviocytes, while also reducing the expression of matrix-degrading enzymes (MMP3, MMP9, ADAMTS5). Importantly, our data extended previous observations by showing that iMac-M2 maintain these antiinflammatory and anti-catabolic effects under sustained inflammatory conditions, including exposure to IL-ip, TNF-a, and LPS. This is a highly important point, as several studies have reported that macrophages, including M2-like populations, may undergo phenotypic drift in inflammatory environments. In contrast to iMac-M2, primary human macrophages exposed to identical stimuli in our study exhibited a pronounced loss of M2 markers and increased expression of Ml -associated markers, highlighting the greater susceptibility of primary macrophages to inflammatory reprogramming. These results indicate that iMac-M2 possess an intrinsic resistance to pro-inflammatory re-education, a property that may be particularly advantageous for therapeutic applications in PTOA, where inflammation is persistent and multifactorial.

[0145] In the DMM-induced PTOA model, a single intra-articular administration of iMac-M2 one week after surgery resulted in measurable improvements in joint function, as evidenced by improved gait symmetry and weight-bearing behavior. These functional4899-6723-7253 2 Page 34 of 52 065472-001005 WOPToutcomes are clinically relevant, as altered gait patterns and load redistribution are strongly associated with pain and disease progression in PTOA.

[0146] At the structural level, iMac-M2 treatment preserved subchondral bone architecture and attenuated synovial inflammation, two early pathological features increasingly recognized as key drivers of PTOA progression. In contrast, effects on cartilage gene expression were comparatively modest, indicaing that iMac-M2 primarily modulate inflammatory and osteo-synovial components rather than directly inducing cartilage regeneration at the analyzed time points. This observation reinforces the importance of early immunomodulatory intervention.

[0147] These findings support the potential of iMac-M2 as an immunomodulatory strategy targeting early inflammatory drivers of PTOA. We also conceive a benefit with repeated intra-articular injections. In addition, combination approaches integrating iMac-M2 with cartilage-protective or regenerative therapies may further enhance therapeutic efficacy.

[0148] Materials and Techniques1. iPSC cell culture and differentiation1.1. iPSC maintenance and expansion

[0149] Human iPSC lines were obtained from the Cedars-Sinai Core facility and were expanded on Matrigel™-coated plates (BD Biosciences) (0.08 mg / well in 6 well plates) and fed every other day with cGMP mTeSR™Plus media (Stymell Technologies). Passaging was performed using ReLeSR™ (StemCell Technologies) following the manufacturer’s protocol.

[0150] Multiple independent human iPSC lines were evaluated during method development. Four lines that reproducibly generated stable iMac-M2 populations were selected for detailed characterization and are presented in this study.1.2. iPSC to iMacrophages differentiation

[0151] iPSC-derived monocytes (iMonocytes) were generated. Briefly, iPSCs were maintained in mTeSRl medium (STEMCELL Technologies, Vancouver, BC, Canada) and differentiated into monocytes following a defined protocol involving cytokine stimulation. For monocyte differentiation, iPSCs were subjected to a sequential protocol involving exposure to the cytokines M-CSF (50 ng / mL, R&D Systems, Minneapolis, MN, USA) and IL-3 (20 ng / mL, R&D Systems) for 7 days in serum-free differentiation medium. The culture medium was changed every 2 days, and cells were maintained at 37°C in a humidified 5% CO2 incubator. The resulting iMonocytes were characterized by flow cytometry, showing expression of monocyte markers (CD14, CDllb).4899-6723-7253 2 Page 35 of 52 065472-001005 WOPT

[0152] iMonocytes were then polarized into M2 macrophages (iMac-M2). Briefly, 1 x 106iMonocytes were incubated in M2 macrophage polarization medium containing IL-4 (20 ng / mL, R&D Systems) and IL-13 (20 ng / mL, R&D Systems) for 48 hours. The medium was replaced every 2 days, and the cells were cultured in a humidified incubator at 37°C with 5% CO2. The differentiated iMac-M2 were characterized by flow cytometry for the expression of macrophage-specific markers (CD206, CD 163). After differentiation, the iMac-M2 were used for cellular injections into the knee joint as part of the in vivo experiment.2. iPSC Characterization2.1. Flow cytometry analysis

[0153] To assess both monocytes and macrophages levels for induction and polarization efficiency, iPSC-derived cells induced the day of harvest, 5- and 7-days post differentiation were lifted with Enzyme (IX) TrypLE™ Express (5 minutes at 37°C) and washed three times with FACS buffer (2% BSA and 0.1% sodium azide in PBS). Cells were either left unstained or stained with anti-human antibody (Table 1), for 20 minutes at 4°C in the dark. After washing, cells were resuspended in FACS buffer, and data were acquired using a BD LSR Fortessa analyzer (BD Biosciences). Gating strategy included exclusion of debris and doublets, followed by live cell gating and marker-specific thresholds defined using fluorescence-minus-one controls. Analysis was performed using FlowJo software (FlowJo LLC).2.2. Phagocytosis Assay with Zymosan Particles

[0154] The functionality of iMac-M2 cells was assessed by phagocytosis using Saccharomyces cerevisiae Zymosan A BioParticles (Alexa Fluor 594 conjugated) (Invitrogen). Zymosan particles were reconstituted in PBS with 2 mM sodium azide and sonicated to disrupt aggregates. Particles were added to adherent iMac-M2 cells at a 2: 1 ratio (particles to cells) in serum -free RPMI medium.

[0155] Phagocytosis was allowed for 30 minutes at 37°C, followed by washing with PBS, trypan blue to quench surface-bound particles, and another PBS wash. Cells were detached with 0.25% trypsin / 0.5 mM EDTA at 4°C for 1 hour, then centrifuged, fixed in 4% formaldehyde, and analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA) to assess the phagocytosis. Phagocytosis was quantified as the percentage of Alexa Fluor 594-positive iMac-M2 cells by flow cytometry after removal of non-intemalized particles during washing steps and quenching of extracellular fluorescence with trypan blue. Data were processed using FlowJo software, with fluorescence-negative cells used to set a threshold for quantifying the percentage of cells that ingested Zymosan.4899-6723-7253 2 Page 36 of 52 065472-001005 WOPT2.3. Bulk RNA sequencing

[0156] Total RNA was extracted from samples using the RNeasy Plus Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA concentration was measured using the Qubit™ RNAHigh Sensitivity Assay Kit (Invitrogen, Carlsbad, CA, USA), and RNA integrity was assessed with the Agilent TapeStation system using RNA ScreenTape (Agilent Technologies, Santa Clara, CA, USA).

[0157] For library preparation, 1000 ng of total RNA per sample (26 pL input volume) was used. Ribosomal RNA depletion was performed using the RiboCop rRNA Depletion Module (Lexogen, Vienna, Austria), followed by library construction with the xGen™ Broad Range RNA Library Prep Kit (Integrated DNA Technologies, IDT, Coralville, IA, USA). Indexing PCR amplification was carried out using the Normalase™ Unique Dual Indexing Primer Plate (IDT, Coralville, IA, USA) with 10 amplification cycles.

[0158] Final libraries were quantified using the Qubit™ dsDNAHigh Sensitivity Assay Kit (Invitrogen, Carlsbad, CA, USA) and assessed for size distribution and quality using the Agilent TapeStation system with High Sensitivity DI 000 ScreenTape (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed on an Illumina NovaSeq platform (Illumina, San Diego, CA, USA) using a 10B flow cell and a 300-cycle sequencing kit, at a loading concentration of 130 pM, with a 1% PhiX control spike-in.

[0159] Raw sequencing reads were processed using a standard RNA sequencing analysis pipeline. Reads were aligned to the rat reference genome (Rnor_6.0), and gene-level read counts were generated. Differential gene expression analysis was performed using the DESeq2 package in R. Adjusted p-values were calculated using the Benjamini-Hochberg method, and genes with an adjusted p-value < 0.05 were considered differentially expressed.3. Coculture of iMac-M2 and hChondrocytes / hSynoviocytes3.1. hChondrocytes and hSynoviocytes isolation

[0160] Human cartilage and synovial tissue samples were obtained from 5 Cedars-Sinai patients undergoing joint replacement surgery, following informed consent. Cartilage tissue was finely minced and digested sequentially with collagenase P 0.1% (w / v) 14h in DMEM / F12 medium at 37°C with agitation to isolate chondrocytes. Synovial tissue was processed separately by digesting with collagenase P 0.1% (w / v) in DMEM / F12 medium under the same conditions to isolate synoviocytes.

[0161] After digestion, cells were filtered through a 70 pm mesh, washed in PBS, and counted. Chondrocytes and synoviocytes were seeded separately in complete culture media (DMEM / F12 supplemented with 10% FBS, 1% penicillin-streptomycin and 0.1% L-ascorbic)4899-6723-7253 2 Page 37 of 52 065472-001005 WOPTat a density of 1 x 106cells per 100 mm plate. Cells were cultured for 48 hours to allow adhesion, followed by biweekly media changes to support growth and maintenance.3.2. Inflammation activation

[0162] Inflammatory media was prepared by adding 10 ng / mL of IL-ip and 100 ng / mL of lipopolysaccharide (LPS) to the chondrocyte and synoviocyte culture media. Chondrocytes were treated with inflammatory media for 24 hours, after which the media was removed for subsequent experiments.3.3. Direct Coculture

[0163] Following 24 hours of inflammation induction, iMac-M2 cells were directly cocultured with chondrocytes. iMac-M2 cells were added at a density of 1 x 104cells per well into wells containing chondrocytes maintained in chondrocyte media.3.4. FACS - Fluorescence Activated Cell sorting

[0164] iMac-M2 cells were stained with a CD14 PE-conjugated mouse monoclonal antibody (anti -human, 1:100 dilution, BioLegend), and then sorted from chondrocytes and synoviocytes at multiple time points (Day 1, Day 3, Day 7, and Day 14) using a BD Influx cell sorter (BD Biosciences) with a 100 pm nozzle, set to the '1.0-drop purify' mode. After sorting, the cells were collected in RLT buffer for gene expression analysis.3.5. Gene expression analysis

[0165] Following FACS, cells were collected, and total RNA was extracted using the RNeasy Plus kit (Qiagen). The RNA was then reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). cDNA was amplified using qPCR with TaqMan® gene expression assays. The threshold cycle (Ct) value for 18S rRNA was used as an internal control, employing the TaqMan® FAM / MGB probe system (4333760F, Thermo Fisher). Gene expression levels were calculated using the Livak method, with fold change determined as 2-AACt, as previously described.4. In vivo injection of iMac-M24.1. Destabilization of Medial Meniscus (DMM)

[0166] A total of 30 immunocompetent male Sprague-Dawley rats (8 weeks old) underwent destabilization of the medial meniscus (DMM) surgery under general anesthesia. Briefly, the procedure involved making a medial parapatellar incision on the right knee to access the joint capsule. Using a microsurgical scalpel, the medial meniscotibial ligament was transected to destabilize the medial meniscus. The controlateral left knee was left unoperated to avoid systemic confounders. Following the procedure, the joint capsule and skin were4899-6723-7253 2 Page 38 of 52 065472-001005 WOPTsutured, and the animals were closely monitored during recovery. Post-operative care included administration of analgesics and ensuring appropriate wound healing to minimize discomfort.4.2. Injection of iMac-M2

[0167] One week after DMM surgery, animals were assigned to receive an intraarticular injection of either iMac-M2 or vehicle control. iMac-M2 cells were harvested, washed twice in sterile phosphate-buffered saline (PBS), and resuspended at the desired concentration immediately prior to injection.

[0168] Rats were anesthetized using isoflurane, and the skin over the knee joint was disinfected. Intra-articular injections were performed under aseptic conditions using a 30-gauge needle inserted through the patellar tendon into the joint space. A total volume of 25 pL containing 1 * 106iMac-M2 cells was injected into the operated knee. Control animals received an equivalent volume of sterile PBS following the same procedure.

[0169] Animals were randomly assigned to treatment groups, and behavioral and histological analyses were performed by investigators blinded to treatment.

[0170] After injection, animals were allowed to recover under close monitoring and were returned to their cages once fully awake. No adverse events related to the injection procedure were observed. Animals were subsequently followed longitudinally for behavioral, imaging, histological, and molecular analyses as described below.5. Serum analysis: IgM ELISA

[0171] In order to assess systemic inflammation markers, blood samples were repetitively collected from each animal at baseline (day of surgery), and at 1-, 2-, 8-, and 16-weeks post-surgery via tail vein puncture. A 0.8 mL serum separator tube (Greiner Bio One MiniCollect, Monroe, NC, USA) was used for collection. The samples were then centrifuged at 2000*g for 10 minutes. Finally, the serum was separated and stored at -80°C until analysis. Serum IgM levels were measured using an ELISA kit (MBS2510638; MyBioSource, San Diego, CA, USA) to evaluate the immune response to iMac-M2 administration. Standard curves were performed in accordance with the standard values indicated by the manufacturer of the ELISA kits. All data sets were exported to Excel, and concentrations were calculated using interpolation in Prism 8 software (GraphPad, La Jolla, CA, USA).6. Analysis of PTO A Development6.1. Behavioral assessment of locomotor function and weight bearing

[0172] Gait analysis was performed to assess functional impairment and disease progression following DMM surgery. Rats were trained to walk along a straight corridor lined with filter paper prior to testing. At baseline and at 48 hours, 2-, 8-, and 16-weeks post-surgery,4899-6723-7253 2 Page 39 of 52 065472-001005 WOPTnon-toxic blue paint was applied to the fore and hind paws, and footprint patterns were collected. Footprints were analyzed to quantify hindlimb gait parameters, including stride length, inter-step distance, and hind paw angle. These measurements were used to assess gait symmetry and limb use between the operated and contralateral hind limbs over time.

[0173] Weight-bearing distribution was assessed to evaluate joint discomfort and functional loading of the hind limbs. Rats were placed in a transparent enclosure and allowed to move freely without restraint. Hindlimb weight distribution was recorded using a pressuresensitive force platform system equipped with high-sensitivity sensors and synchronized overhead video monitoring. Weight distribution between the operated and contralateral hind limbs was continuously recorded over a 5 minutes period and used as an index of joint incapacitance and pain-related behavior.6.2. X-ray microtomography analysis of subchondral bone

[0174] In-vivo micro computed tomography (pCT) scans were used to assess the bone mineral density (BMD) and structural parameters of the knee joints. Scans were performed using a Quantum GX2 microCT scanner (PerkinElmer, Waltham, MA, USA) at a voltage of 55kVp and a current of 145mA, with an integration time of 200ms and a voxel size of 35pm. The entire knee joint was scanned to capture structural details of the bone. Analysis Software to contour the region of interest and quantify key architectural parameters including: bone volume, total volume, bone volume fraction (BV / TV), and mean density. These parameters were used to evaluate the effects of iMac-M2 administration on the progression of post-traumatic osteoarthritis (PTOA) in the DMM rat model.

[0175] Image reconstruction and quantitative analyses were performed using Micro View software (Parallax Innovations Inc., Ilderton, ON, Canada).6.3. Histology and OARSI scoring

[0176] Following sacrifice, the knee joints were harvested, fixed in 4% formaldehyde, dehydrated through graded ethanol, and embedded in paraffin. Five-micron-thick sections were cut from the paraffin blocks.

[0177] Sections were stained with hematoxylin and eosin (H&E) to assess overall tissue morphology and synovial architecture. Safranin O / Fast Green staining was performed to evaluate cartilage integrity and glycosaminoglycan (GAG) content.

[0178] Cartilage degeneration was assessed using the OARSI scoring system on Safranin O / Fast Green-stained sections. Scoring was performed on X sections per joint by X observers blinded to treatment.4899-6723-7253 2 Page 40 of 52 065472-001005 WOPT

[0179] Synovial vascularization was quantified on H&E-stained sections. Vascular density was assessed by manually counting blood vessels within the synovial tissue using QuPath software (version 0.5.0, University of Edinburgh, Edinburgh, UK). Quantification was performed on standardized regions of interest from multiple sections per joint by investigators blinded to treatment, and vascular density was expressed as the number of vessels per mm2.7. Analysis of pain7.1. Von Frey.

[0180] Pain is a key clinical feature of PTOA and is often associated with inflammatory signaling, aberrant joint innervation, and central sensitization. To explore iMac-M2 therapy for mitigating pain sensitivity, we integrated behavioral testing with molecular and histological analyses. Pain-related behavior was assessed using von Frey (VF) filament testing throughout the post-surgical timelineExample 2. Production and polarization from iPSC to iMac-M2.Table 1. Exemplary differentiation protocol for iPSCs into functional iMac-M2:4899-6723-7253 2 Page 41 of 52 065472-001005WOPTCell Culture Setup:1- Grow iPSCs on Matrigel (Coming) in mTeSR (StemCell Technologies Inc.) until 80% confluency is reached.2- Passage cells using StemPro EZPassage Disposable Stem Cell Passaging Tool (Life Technologies) and plate on Matrigel in mTeSR.iPSC Cell Maintenance and Selection:3- Maintain cells over the next 2-4 days, removing differentiating cells or abnormally shaped ones.4- Wait until wells have approximately 10-12 colonies measuring 1.0 mm in diameter.Initiate Differentiation (Stage 1): To obtain Embryoid Bodies (EBs)5- Start differentiation using stage 1 media (80ng / ml BMP4 in mTeSR).6- Change media daily for 4 days.• Properly formed EBs usually appear as spherical or spheroid-like, three- dimensional aggregates, which are relatively uniform in size.• To verify the pluripotent nature of EBs and confirm their capacity to differentiate into the three embryonic germ layers, specific genes or proteins can be examined such as:o Ectoderm: Nestin (early neuroectoderm), pill-tubulin (TUJ1), and Soxl (related to neurogenesis),o Mesoderm: Brachyury (T), Desmin, and a-SMA (smooth muscle actin), ando Endoderm: AFP (alpha-fetoprotein), Sox 17, and GATA.Stage 2 and Stage 3: To obtain myeoloid progenitors (iMyeloid progenitor)Stage 2:7- Switch cells to stage 2 media (25ng / ml bFGF, lOOng / ml SCF, and 80ng / ml VEGF) in StemPro-34 serum-free medium (Gibco).• At this stage we have a mix of population in the myeloid lineage. As seen in Stage 4, monocyte is a specific cell of the myeloid lineage, and monocyte has4899-6723-7253 2 Page 42 of 52 065472-001005WOPTthe specificity to float and the others cells will be adherent, a difference in property which may be utilized for purification or separation.• When transitioning from EBs to iMonocyte progenitors derived from iPSC- derived hematopoietic precursors, key markers have changes that may be analyzed via immunostaining or flow cytometry to verify commitment to the monocyte lineage:o CD14: monocyte marker, which is increased compared to EBs;o CDllb: increases as cells progress along the myeloid / monocytic path and lose their more primitive, EB-like features;o CD115 (CSF1R): increases with the development of monocyte / macrophage progenitors, indicating a shift toward functional myeloid cells; ando CD34: decreases relative to EBs; originally associated with early hematopoietic progenitors, CD34 expression wanes as cells commit to the monocyte lineage.Stage 3 :8- Two days later, switch cells to stage 3 media (StemPro-34 supplemented with 50ng / ml SCF, 50ng / ml IL-3, 5ng / ml TPO, 50ng / mL M-CSF, and 50ng / mL Flt3).Stage 4 (Day 14): to obtain matures iMonocytes9- switch cells to stage 4 media (50ng / ml M-CSF, 50ng / ml Flt3 ligand, and 25ng / ml GM- CSF in StemPro-34 medium).10- Day 18, collect floating cells in the supernatant, pellet, resuspend in fresh stage 4 media, and distribute evenly among wells.Cell Sorting and Expansion Day 22: Differentiation into iMacrophages-MO (Non polarized) 11- sort floating cells for CD14+ cells.12- Plate sorted cells at a density of 50* 103cells / cm2in RPMI-1640 medium supplemented with 2mM GlutaMAX, lOng / ml GM-CSF, and lOOng / ml IL-34. (The RPMI-1640 medium is supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic.) 13- Perform half-media changes every other day for 2 weeks.GlutaMax is formulated as a 200 mM (100*) L-alanyl-L-glutamine dipeptide in 0.85% NaCl. It is a substitute for L-glutamine.Collection:4899-6723-7253 2 Page 43 of 52 065472-001005 WOPT14 - Day 35 - 40 collect cells and inject in vivo in 25 .1 PBS15 - iMacs-M2 will be FACS-sorted to generate a pure population of CD163+ / CD206+ cells.Polarization:16- In RPMI medium supplemented with 10% FBS, 1% antibiotic-antimycotic (A / A), and 20 ng / mL of M-CSF, IL-4, IL-6, and IL-13, cells are cultured for M2 differentiation. After 2 days of differentiation, the cells can be collected and used, but their M2 characteristics may diminish by day 14 after collection.

[0181] Evaluation criteria of iMac-M2:1. Flow cytometry analysis<Control negative: pluripotent stem cell marker Tra-1-60 and the granulocyte marker CD66b.2. Phagocytosis assay: via Cayman’s phagocytosis assay kit (IgG PE).3. Migration assay / Boyden cell migration assay: to evaluate the ability of iMac-M2 macrophages to migrate towards specific chemokine factors.

[0182] Specifically, procedures in the migration assay include:(1). Cell Preparation:a. Trypsinization of cells.b. Incubate cells in starvation media (media with 5% FBS) for 1 hour.c- Cell Resuspension: Resuspend cells in DMEM at a concentration of 5 * 105cells / mL. d- Filtration: Use polycarbonate PFB filters (Neuro Probe) with 8pm pores.(2). Chamber Preparation:a- Place culture media with 20 ng / ml of TNF-a or 100 ng / ml MCP1 in the bottom chamber of Neuro Probe 48-well chemotaxis chambers (Neuro Probe).b- Cell Seeding: Seed 5 * 104cells in the top chamber.c- Incubation: Incubate at 37°C in a 95% 02 / 5% CO2 environment for 24 hours. (3). Cell Fixation:a. Fix migrated cells in methanol.b. Staining: Stain fixed cells with 500 nM DAPI.(4). Analysis: Fluorescence microscopy imaging -> yes or no response.4899-6723-7253 2 Page 44 of 52 065472-001005 WOPT

[0183] In vivo evaluations:

[0184] An exemplary in vivo administration of iMac-M2 in immunocompetent rats following DMM surgery is shown in figure 3 A.

[0185] Behavioral tests: a baseline (before injury) is chosen. (1) Gait Analysis: to assess disease progression by following stride length, inter-step length and hind paw angle. Rats will be trained to walk on filter paper before the test. For testing at baseline or at 48h, 2-, 8- and 16-weeks post-injury, blue nontoxic paint will be applied to the hind and fore paws. The resulting footprint traces will be analyzed, measuring (i) stride length for hindlimbs, determined by measuring the average distance of forward movement between each stride; (ii) inter-step distance by measuring the distance between steps for both hindlimbs; and (iii) hind paw angle by measuring the relative angle between the distal portion of the thenal pad and the proximal portion of the heel during forward movement. (2) Von Frey Test: to assess mechanical and tactile sensitivity related to pain. Using filaments that range from 0.6 to 15 g, rats will be poked and accessed for a response. The 4 will be the baseline filament, in which a response will mean to go to a smaller filament, and no response will lead to a larger filament, Responses depend on behavioral associated to test paw. A Von Frey gird from Up Down reader will be used for data acquisition and Up Down Reader software will be used to analyze the data. (3) Weight bearing Test: to assess changes in incapacitance or weight distribution in both hind paws without the need to restrain the animal. Rats are placed into a clear cage and allowed to freely move. The system employs a combination of 2000 high-sensitivity force sensors that measure the rat’ s weight distribution on four paws and video monitoring from above in order to precisely measure and monitor in real-time changes in their postural equilibrium. Rats are typically monitored for 5 minutes.

[0186] Blood collection to assess level of inflammation and rejection of iMac-M2: First time point (Day 0) IS before surgery (baseline); Then after surgery (week 1), after 2-, 8- and 16-weeks, to perform ELISA to test IgM and serum cytokine levels of IL-ip, IL-6 and TNF-a. Prior to analysis of cytokine expression total protein contents will be quantified using a BCA protein assay kit (ThermoFisher). ELISAs detecting rat IL-6, IL-ip, tumor necrosis factor (TNF)-a and CTX-II (Quantikine ELISA Kit, R&D systems)

[0187] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless4899-6723-7253 2 Page 45 of 52 065472-001005 WOPTspecifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0188] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0189] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of’ or “consisting essentially of.”4899-6723-7253 2 Page 46 of 52 065472-001005 WOPT

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for treatingjoint injury, reducing inflammation in the joint, or inhibiting post- traumatic osteoarthritis in an individual in need thereof, comprising: administering a therapeutically effective quantity of M2 phenotype-polarized macrophages derived from induced pluripotent stem cells (iMac-M2) to the individual.

2. The method of claim 1, wherein the iMac-M2 continue to express CD14, CD68, and CD 163 for at least 34 days, at least 19 days, at least 16 days, at least 13 days, or at least 7 days after derivation from the iPSC.

3. The method of claim 1, wherein the iMac-M2 are administered locally to the joint or to a site of osteoarthritis, and wherein blood IgM level is not increased in response to the administration.

4. The method of claim 1, wherein the method reduces IL6, ILip, and / or IL8 levels in the joint or in inflamed chondrocytes and / or inflamed synoviocytes, optionally the inflamed chondrocytes and / or the inflamed synoviocytes having been obtained from a patient with osteoarthritis.

5. The method of claim 1, wherein the method reduces ADAMTS5, MMP3, MMP9, and / or MMP13 levels in the joint or in inflamed chondrocytes and / or inflamed synoviocytes, optionally the inflamed chondrocytes and / or the inflamed synoviocytes having been obtained from a patient with osteoarthritis.

6. The method of claim 1, wherein the iMac-M2 are administered before osteoarthritis is diagnosed in the individual or within 10 days of the injury.

7. The method of claim 1, wherein the individual has a joint injury comprising a fracture, a ligament tear, or a meniscal damage, or the individual has a tendon inflammation.

8. The method of claim 1, wherein the joint comprises a knee joint, a finger joint, a toe joint, a wrist joint, an ankle joint, an elbow joint, a hip joint, a should joint, a pivot joint, a joint between the vertebrae, or a joint where a thumb joins a hand.

9. The method of claim 1, wherein the individual is a human.

10. The method of claim 1, wherein the iMac-M2 are produced by a process comprising:a. incubating isolated mononuclear phagocytes derived from pluripotent stem cells with a first culture media comprising macrophage colony-stimulating factor (M-CSF), wherein the mononuclear phagocytes derived from pluripotent stem cells differentiate into cells of macrophage lineage which express CD68;4899-6723-7253 2 Page 47 of 52 065472-001005 WOPTb. incubating the cells of the macrophage lineage with a second culture media comprising M-CSF, IL-4, IL-6, and IL-13, wherein the cells of the macrophage lineage is polarized to an M2 phenotype; andc. recovering the cells of the macrophages lineage polarized to the M2 phenotype, wherein the cells polarized to the M2 phenotype co-express CD 163 and CD206.

11. The method of claim 10, wherein the process of producing the iMac-M2 further comprises steps (i-v), before the step (a), comprising:i. incubating induced pluripotent stem cells with a third culture media comprising bone morphogenetic protein (BMP-4) to obtain cells in the form of embryoid bodies; ii. incubating the cells of the form of embryoid bodies with a first serum-free culture media comprising basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stem cell factor (SCF) for two days;iii. incubating the cells after step (ii) with a second serum-free culture media comprising SCF, IL-3, thrombopoietin (TPO), macrophage colony-stimulating factor (M-CSF), and Fms-like tyrosine kinase 3 ligand (FLT3 ligand) for 6-8 days;iv. incubating the cells after step (iii) with a third serum-free culture media comprising FLT3 ligand, GM-CSF, and M-CSF, thereby obtaining mononuclear phagocytes derived from pluripotent stem cells; andv. recovering the mononuclear phagocytes after step (iv), thereby obtaining isolated mononuclear phagocytes derived from pluripotent stem cells,wherein the mononuclear phagocytes derived from pluripotent stem cells express CD14.

12. A method for obtaining macrophages polarized to an M2 phenotype from pluripotent stem cells, the method comprising:a. incubating isolated mononuclear phagocytes derived from pluripotent stem cells with a first culture media comprising macrophage colony-stimulating factor (M-CSF), wherein the mononuclear phagocytes derived from pluripotent stem cells differentiate into cells of macrophage lineage which express CD68;b. incubating the cells of the macrophage lineage with a second culture media comprising M-CSF, IL-4, IL-6, and IL-13, wherein the cells of the macrophage lineage is polarized to an M2 phenotype; and c. recovering the cells of the macrophages lineage polarized to the M2 phenotype, wherein the cells after step (c) co-express CD 163 and CD206.4899-6723-7253 2 Page 48 of 52 065472-001005 WOPT13. The method of claim 12, which comprises additional steps (i-v), before the step (a), comprising:i. incubating induced pluripotent stem cells with a third culture media comprising bone morphogenetic protein (BMP-4) to obtain cells in the form of embryoid bodies; ii. incubating the cells of the form of embryoid bodies with a first serum-free culture media comprising basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stem cell factor (SCF);iii. incubating the cells after step (ii) with a second serum-free culture media comprising SCF, IL-3, thrombopoietin (TPO), macrophage colony-stimulating factor (M-CSF), and Fms-like tyrosine kinase 3 ligand (FLT3 ligand);iv. incubating the cells after step (iii) with a third serum-free culture media comprising M-CSF, GM-CSF, and FLT3 ligand, thereby obtaining mononuclear phagocytes derived from pluripotent stem cells; andv. recovering the mononuclear phagocytes after step (iv), thereby obtaining isolated mononuclear phagocytes derived from pluripotent stem cells,wherein the mononuclear phagocytes derived from pluripotent stem cells express CD14.

14. The method of claim 13, wherein the recovering of the step (v) comprises collecting floating cells every 3-5 days or 2 times per week from the incubation of step (iv), repeatedly for a time span of about 4-70 days.

15. The method of claim 13, wherein the step (i) comprises the BMP4 at about 60-100 ng / mL; the step (ii) comprises the bFGF at about 20-30 ng / mL, the SCF at about 80- 120 ng / mL, and the VEGF at about 60-100 ng / mL; the step (iii) comprises the SCF at about 40-60 ng / mL, the IL-3 at about 10-60 ng / mL, the TPO at about 1-10 ng / mL, the M-CSF at about 40-60 ng / mL, and the FLT3 ligand at about 40-60 ng / mL; the step (iv) comprises the M-CSF at about 40-60 ng / mL, the FLT3 ligand at about 40-60 ng / mL, the GM-CSF at about 20-30 ng / mL.

16. The method of claim 13, wherein the step (i) comprises the BMP4 at about 80 ng / mL;the step (ii) comprises the bFGF at about 25 ng / mL, the SCF at about 100 ng / mL, and the VEGF at about 80 ng / mL; the step (iii) comprises the SCF at about 50 ng / mL, the IL-3 at about 20 ng / mL, the TPO at about 5 ng / mL, the M-CSF at about 50 ng / mL, and the FLT3 ligand at about 50 ng / mL; the step (iv) comprises the M-CSF at about 50 ng / mL, the FLT3 ligand at about 50 ng / mL, and the GM-CSF at about 25 ng / mL.4899-6723-7253 2 Page 49 of 52 065472-001005 WOPT17. The method of claim 13, wherein the step (i) includes the incubating with the third culture media comprising the BMP -4 for about 4 days; the steps (ii) and (iii) include the incubating with the first serum-free and the incubating with the second serum-free media for a total of about 14 days; and the step (iv) includes the incubating with the third serum-free culture media for at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, or at least 3 months.

18. The method of claim 12, wherein the step (a) includes the incubating for about 5 days or from 4 to 7 days with the first culture media comprising the M-CSF at about 20 ng / mL or from 5 to 50 ng / mL; and the step (b) includes the incubating for about 2 days or from 1 to 5 days with the second culture media comprising the M-CSF, the IL-4, the IL-6, and the IL-13, each at about 20 ng / mL or from 5 to 50 ng / mL.

19. A composition comprising a quantity of M2 phenotype-polarized macrophages derived from induced pluripotent stem cells (iPSC), wherein the macrophages continue to express CD14, CD68, and CD163 for at least 34 days after derivation from the iPSC, wherein the macrophages are derived in a process comprising:a. incubating isolated mononuclear phagocytes derived from pluripotent stem cells with a first culture media comprising macrophage colony-stimulating factor (M-CSF), wherein the mononuclear phagocytes derived from pluripotent stem cells differentiate into cells of macrophage lineage which express CD68;b. incubating the cells of the macrophage lineage with a second culture media comprising M-CSF, IL-4, IL-6, and IL-13, wherein the cells of the macrophage lineage is polarized to an M2 phenotype; andc. recovering the cells of the macrophages lineage polarized to the M2 phenotype, wherein the cells polarized to the M2 phenotype co-express CD 163 and CD206.

20. The composition of claim 19, wherein the process further comprises steps (i)-(v), before the step (a), wherein the steps (i)-(v) comprise:i. incubating induced pluripotent stem cells with a third culture media comprising bone morphogenetic protein (BMP-4) to obtain cells in the form of embryoid bodies; ii. incubating the cells of the form of embryoid bodies with a first serum-free culture media comprising basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and stem cell factor (SCF) for two days;4899-6723-7253 2 Page 50 of 52 065472-001005 WOPTiii. incubating the cells after step (ii) with a second serum-free culture media comprising SCF, IL-3, thrombopoietin (TPO), macrophage colony-stimulating factor (M-CSF), and Fms-like tyrosine kinase 3 ligand (FLT3 ligand) for 6-8 days;iv. incubating the cells after step (iii) with a third serum-free culture media comprising FLT3 ligand, GM-CSF, and M-CSF, thereby obtaining mononuclear phagocytes derived from pluripotent stem cells; andv. harvesting the mononuclear phagocytes repeatedly every 3-5 days for at least 3 weeks, 6 weeks, 9 weeks, or 12 weeks;wherein the mononuclear phagocytes derived from pluripotent stem cells express CD14.4899-6723-7253 2 Page 51 of 52 065472-001005 WOPT

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