Generation, purification and use of lipocartilage cells comprising lipid vacuoles

Abstract

The present application relates to lipid vacuole (aka LV)-containing lipocartilage cells (aka LCs), and methods for the differentiation of human lipocartilage progenitors into LV-containing LCs. Also disclosed are methods related to purifying, labeling, maturing, and delivering of LV-containing LCs to a subject in need thereof.

Description

UCI029.002WO PCT APPLICATIONGENERATION, PURIFICATION AND USE OF LIPOCARTILAGE CELLSCOMPRISING LIPID VACUOLESRELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims the benefit of U.S. Provisional Ser. No. 63 / 737600, filed December 20, 2024, which is hereby incorporated by reference in its entirety as if fully set forth herein.RELATED FIELD

[0002] Disclosed herein are super-stable lipid vacuole (aka LV)-containing lipocartilage cells (LCs), and their usage thereof. The present disclosure also relates to methods of differentiating human stem cells into LCs, and the labeling of their LVs, purifying LCs based on their labeled LVs, maturing purified LCs, and delivering of LCs to a subject in need thereof.BACKGROUND

[0003] Cartilage serves as an important component of the vertebrate skeletal system, playing both structural and mechanical roles. Tissues similar to vertebrate cartilage also exist in multiple invertebrate groups, including annelids, cephalopods, and horseshoe crabs. Conventionally, differentiated (aka mature) cells of the cartilage, called chondrocytes, secrete large quantities of extracellular matrix (ECM), which endows cartilage tissue with its shape and mechanical properties. During embryogenesis, cartilages arise both from mesodermal and neural crest sources, and, in some cranial cartilage, cells from these two embryonic origins can join together.SUMMARY OF SOME EXAMPLE EMBODIMENTS

[0004] Disclosed herein is lipid-laden cartilage (henceforth lipocartilage) consisting of vacuolated cells called LCs, methods of preparing, and uses thereof. Some of the embodiments of the present disclosure relate to a method of selecting for adult and / or differentiated cartilage cells among a population of cells by screening for the presence of LVs.Some embodiments relate to methods for the in vitro differentiation of human cartilage progenitor cells (henceforth progenitors) into LV-containing LCs. Also disclosed herein are methods to methods to label LVs in LCs, purify labeled LCs, and methods to deliver purified LCs to a subject in need thereof. In some embodiments, LC progenitors are sourced from autologous ear cartilage, autologous nasal cartilage, autologous rib cartilage, autologous xiphoid cartilage, autologous articular cartilage, or any combination thereof. In some embodiments, LC progenitors are produced by inducing cartilage identity in non-cartilage mesenchymal cells, such as adipose tissue-derived mesenchymal stem cells. In some embodiments, cartilage progenitors a produced by inducing cartilage identity in non-cartilage mesenchymal cells such as adipose tissue-derived mesenchymal stem cells. In some embodiments, LC progenitors are produced by inducing cartilage identity in non-cartilage mesenchymal cells, such as bone marrow-derived mesenchymal stem cells. In some embodiments, LC progenitors are produced by inducing cartilage identity in non-cartilage mesenchymal cells, such as peripheral blood-derived mesenchymal stem cells. In some embodiments, adipose tissue-derived mesenchymal stem cells are expanded prior to LC progenitor induction using a bioreactor, spinner flask, roller bottle, multilayered flask, or any combination thereof. In some embodiments, bone marrow-derived mesenchymal stem cells are expanded prior to LC progenitor induction using a bioreactor, spinner flask, roller bottle, multilayered flask, or any combination thereof. In some embodiments, peripheral blood- derived mesenchymal stem cells are expanded prior to LC progenitor induction using a bioreactor, spinner flask, roller bottle, multilayered flask, or any combination thereof. In some embodiments, LC progenitors are induced to differentiate into LCs by means of chondrogenic culture medium (CCM), containing DMEM with high glucose / GlutaMAX, Penicillin- Streptomycin-Fungizone, non-essential aminoacids, L-proline, sodium pyruvate, and dexamethasone, and supplemented with fetal bovine serum, TGF-01, bFGF, and PDGF. In some embodiments, CCM further comprises additionally supplemented BMP1, BMP2, BMP5, GDF5, GDF10, IGF1, IGF2, IGFBP2, IGFBP4, IGFBP5, IGFBP6, IGFBP7, TGFB3, DKK3, SFRP1, SFRP2, SFRP5, WIFI, ANGPT4, ANGPTL7, FGF7, FGF18, CYTL1, or any combination thereof. In some embodiments, mature LCs are selected based on the presence of LVs. In some embodiments, mature LCs are labeled with lipid-binding dyes BODIPY 493 / 503, BODIPY 500 / 510, BODIPY 530 / 530, BODIPY 558 / 568, BODIPY 576 / 589, BODIPY581 / 591, LipiDye, LipidGreen, LipidGreen2, LipidTox, SMCy3, SMCy3.5, SMCy5, SMCy5.5, SMCy7, SMCy7.5, or any combination thereof. In some embodiments, mature LV- containing LCs are purified via standard centrifugation based on being more buoyant than non- LV containing cells. In some embodiments, fluorescently labeled live LCs are purified via standard fluorescent activated cell sorting (FACS) approach based on being fluorescent lipid dye-positive. In some embodiments, LCs are matured in media in the presence of insulin and / or IGF1.

[0005] Also disclosed herein is a composition. In some embodiments, the composition has use in making a synthetic extracellular matrix (ECM) gel. In some embodiments, the ECM gel comprises an at least one protein selected from: Collagen VI, Collagen VII, Collagen VIII, Collagen IX, Collagen X, Collagen XI, Myocilin, Fibronectin, Aggrecan, Tenascin, Thrombospondin, or any combination thereof. In some embodiments, the abundance of the at least one protein mimics the protein abundance profile observed in head and neck cartilage ECM. In some embodiments, the composition is combined with chitosan to generate a protein-chitosan ECM construct. In some embodiments, the composition is prepared in any custom shape.

[0006] Also disclosed herein is a method to generate LC-containing living-tissue construct (henceforth tissue construct). In some embodiments, purified LCs are seeded onto a ECM composition of any embodiment of the present disclosure, and maintained in vitro. In some embodiments, tissue construct is grafted onto a subject. In some embodiments, tissue construct comprises an at least one LC in suspension. In some embodiments, tissue construct comprises a LC pellet. In some embodiments, the subject is mammalian and / or human. In some embodiments, the subject requires reconstructive surgery of cartilages of head (nose or ear cartilage) and / or neck (larynx, trachea). In some embodiments, the method results in a natural replacement for lost cartilage elements of the head and neck. In some embodiments, LCs or LC-containing constructs have function as structural support of non-cartilage craniofacial tissues, including skin and muscle. In some embodiments, the subject requires replacement or reconstructive surgery of intervertebral discs. In some embodiments, the method produces a natural replacement for damaged intervertebral discs.

[0007] Also disclosed herein is a method of generating an adult lipocartilage cell, the method comprising incubating a progenitor cell capable of differentiating into the adultlipocartil age cell with cartilage-inducing media for at least 20 days. In some embodiments, the progenitor cell is a stem cell, optionally wherein the progenitor cell is a pluripotent and / or induced pluripotent stem cell. In some embodiments, the progenitor cell is incubated with cartilage-inducing media for at least 25, 30, 35, 40, 45, 50, 55, 60, or 65 days. In some embodiments, the progenitor cell is continuously shaken and / or swirled during incubation. In some embodiments, the cartilage-inducing media is chondrogenic culture and / or differentiation media, optionally wherein the cartilage-inducing media is MesenCult™-ACF Chondrogenic Differentiation Medium for MSCs. In some embodiments, the chondrogenic culture and / or differentiation media comprises at least one of: DMEM, high glucose / GlutaMAX, Penicillin, Streptomycin, Fungizone, an at least one amino acid, L- proline, pyruvate, dexamethasone, fetal bovine serum, TGF-bl, bFGF, PDGF, or any combination thereof. In some embodiments, the chondrogenic culture and / or differentiation media comprises at least one of: BMP1, BMP2, BMP5, GDF5, GDF10, IGF1, IGF2, IGFBP2, IGFBP4, IGFBP5, IGFBP6, IGFBP7, TGFB3, DKK3, SFRP1, SFRP2, SFRP5, WIFI, ANGPT4, ANGPTL7, FGF7, FGF18, CYTL1, or any combination thereof. In some embodiments, the progenitor cell originates from autologous ear cartilage, autologous nasal cartilage, autologous rib cartilage, autologous xiphoid cartilage, autologous articular cartilage, non-cartilage mesenchymal cells, adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, or any combination thereof.

[0008] Also disclosed herein is a method of identifying an adult lipocartilage cell from a population of cells. In some embodiments, the method comprises screening for the presence of a lipid droplet (also referred to as a lipid vacuole, or “LV”) within a cell, wherein the presence of a lipid droplet indicates that the cell is the adult lipocartilage cell. In some embodiments, the screening is performed through fluorescent activated cell sorting (FACs). In some embodiments, the screening is performed through a buoyancy assay, wherein the presence of the lipid droplet in the adult lipocartilage cell results in a different cellular buoyancy compared with other cells in the population of cells. In some embodiments, the buoyancy assay is centrifugation. In some embodiments, the presence of the lipid droplet is determined by positive staining from a dye. In some embodiments, the dye is a fluorescent lipid dye. In some embodiments, the dye is a BODIPY dye. In some embodiments, the dye isselected from: BODIPY 493 / 503, BODIPY 500 / 510, BODIPY 530 / 530, BODIPY 558 / 568, BODIPY 576 / 589, BODIPY 581 / 591, LipiDye, LipidGreen, LipidGreen2, LipidTox, SMCy3, SMCy3.5, SMCy5, SMCy5.5, SMCy7, SMCy7.5, or any combination thereof.

[0009] Also disclosed herein is a method of purifying an adult lipocartilage cell from a population of cells. In some embodiments, the method comprises: screening for the presence of a lipid droplet within a cell; and purifying the cell comprising the lipid droplet. In some embodiments, the screening is performed through fluorescent activated cell sorting (FACs). In some embodiments, the screening is performed through a buoyancy assay, wherein the presence of the lipid droplet in the adult lipocartilage cell results in a different cellular buoyancy compared with other cells in the population of cells. In some embodiments, the buoyancy assay is centrifugation. In some embodiments, the presence of the lipid droplet is determined by positive staining from a dye. In some embodiments, the dye is a fluorescent lipid dye. In some embodiments, the dye is a BODIPY dye. In some embodiments, the dye is selected from: BODIPY 493 / 503, BODIPY 500 / 510, BODIPY 530 / 530, BODIPY 558 / 568, BODIPY 576 / 589, BODIPY 581 / 591, LipiDye, LipidGreen, LipidGreen2, LipidTox, SMCy3, SMCy3.5, SMCy5, SMCy5.5, SMCy7, SMCy7.5, or any combination thereof.

[0010] Also disclosed herein is a method of staining an adult lipocartilage cell. In some embodiments, the method comprises administering to the adult lipocartilage cell a dye specific for a lipid droplet. In some embodiments, the dye is a fluorescent lipid dye. In some embodiments, the dye is a BODIPY dye. In some embodiments, the dye is selected from: BODIPY 493 / 503, BODIPY 500 / 510, BODIPY 530 / 530, BODIPY 558 / 568, BODIPY 576 / 589, BODIPY 581 / 591, LipiDye, LipidGreen, LipidGreen2, LipidTox, SMCy3, SMCy3.5, SMCy5, SMCy5.5, SMCy7, SMCy7.5, or any combination thereof.

[0011] Also disclosed herein is a method of generating a population of adult lipocartilage cells. In some embodiments, the method comprises: screening a general population of cells for the presence of a lipid droplet within a cell; purifying the cell comprising the lipid droplet, thus isolating an adult lipocartilage cell; and expanding the adult lipocartilage cell, thus forming the population of adult lipocartilage cells. In some embodiments, the expansion is performed by incubating the adult lipocartilage cell in the same media that the general population of cells was first grown in. In some embodiments, the expansion is performed for up to two weeks. In some embodiments, the expansion comprisesadministering a media with insulin, IGF1, BMP1, BMP2, glucose, or any combination thereof. In some embodiments, the glucose is present at about 5 mM, 7 mM, 10 mM, 15 mM, 25 mM, or any integer that is between about 5 and about 25 mM. In some embodiments, the expansion is performed in a bioreactor, spinner flask, roller bottle, multilayered flask, or any combination thereof.

[0012] Also disclosed herein is a composition for use as a synthetic extracellular matrix (ECM) gel, the composition comprising at least one of: Collagen VI, Collagen VII, Collagen VIII, Collagen IX, Collagen X, Collagen XI, Myocilin, Fibronectin, Aggrecan, Tenascin, Thrombospondin, or any combination thereof. In some embodiments, the composition further comprises chitosan.

[0013] Also disclosed herein is a method of generating a replacement tissue construct. In some embodiments, the method comprising seeding the population of adult lipocartilage cells generated from the method of any one of the embodiments of the present disclosure, with the composition of any one of the embodiments of the present disclosure.

[0014] Also disclosed herein is a replacement tissue construct comprising the lipid droplet-containing adult lipocartilage cell generated using the method of any one of the embodiments of the present disclosure. In some embodiments, the replacement tissue construct has use for grafting onto a subject. In some embodiments, the replacement tissue construct has use as part of replacement and / or reconstructive surgery in a subject in need thereof. In some embodiments, the subject is mammalian and / or human. In some embodiments, the subject has a head and / or a neck cartilage defect. In some embodiments, the head and / or neck cartilage defect is a defect in nose, ear, laryngeal, or tracheal cartilage, or any combination thereof. In some embodiments, the replacement tissue construct provides structural support of noncartilage craniofacial tissues in the subject. In some embodiments, the non-cartilage craniofacial tissue is skin and / or muscle. In some embodiments, the subject requires replacement and / or reconstructive surgery of intervertebral discs.

[0015] Also disclosed herein is a kit for use in tissue grafting, the kit comprising the adult lipocartilage cell generated using the method of any one of the embodiments of the present disclosure.

[0016] Also disclosed herein is a method of grafting the adult lipocartilage cell generated using the method of any one of the embodiments of the present disclosure into asubject in need thereof, the method comprising administering the adult lipocartilage cell as part of a suspension.

[0017] Also disclosed herein is a method of grafting the adult lipocartilage cell generated using the method of any one of the embodiments of the present disclosure into a subject in need thereof, the method comprising administering the adult lipocartilage cell as part of a cell pellet.

[0018] Also disclosed herein is a method of grafting the adult lipocartilage cell generated using the method of any one of the embodiments of the present disclosure into a subject in need thereof, the method comprising administering the adult lipocartilage cell as part of a custom- shaped artificial extracellular matrix seeded with lipocartilage cells.BRIEF DESCRIPTION OF THE FIGURES

[0019] FIGs. 1A-1C depict a nonlimiting example of LCs present in multiple cartilages that impart biomechanical properties. (FIG. 1A) Schematic drawing of mouse cartilages color-coded by embryonic origin. LC-containing neural crest (NC)-derived lipocartilage shown in green. (FIG. IB) Biomechanical analyses of mouse ear lipocartilage (Ear) compared to white adipose tissue (WAT). Despite morphological similarities, lipocartilage displays significantly higher stiffness (Young’s modulus; left; 3.25±0.38 MPa; Welch’s t test, p=0.0004), ultimate tensile strength (UTS; center; 1.30±0.24 MPa; Welch’s t test, p=0.005), than WAT, but lower strain at failure (SAF; right; 0.60±0.10, p=0.0273). (FIG. 1C) Biomechanical analyses of native rat ear lipocartilage (control) and delipified tissue. Compared to control (green), stiffness (left; 3.27±0.52 vs. 5.79±0.99 MPa; Welch’s t test, p=0.0004), UTS (center; 1.91±0.22 v . 4.53±0.92 MPa; Welch’s t test, p=0.0259), and resilience (right; 0.38±0.06 vs. 1.77±0.48 MJm'3; Welch’s t test, p=0.0228) increase significantly following delipification (gray). Clonogenic analysis in Col2al-CreER ;R26R mice shows lacZ-labeled LCs form discrete clones that are stable in size for six months (Kolmogorov-Smirnov test, p=0.506).

[0020] FIGs. 2A-2L depicts a nonlimiting example of human head and neck cartilage accumulating LVs. (FIG. 2A) Alcian blue staining of human fetal ear cartilage at gestation week (GW) 20-21. (FIG. 2B) OilRedO staining pattern in GW20-21 human ear. Numerous LVs are present in chondrocytes (as shown on the right panel with arrows). (FIGs.2C-2F) Electron micrographs of GW20-21 human thyroid cartilage (FIG. 2C), epiglottis (FIG. 2D), ear cartilage (FIG. 2E), and nasal cartilage fold (FIG. 2F) reveal chondrocytes with large LVs. (FIG. 2G) Alcian blue staining of hESC-derived cartilage pellet at in vitro culture day 65. (FIGs. 2H-I) OilRedO staining of day 65 pellets shows numerous LVs in chondrocytes. (FIG. 2J) Label-free imaging of day 65 cartilage pellets reveals LVs (SRS signal) and fibrillar collagen (second harmonics generation signal). (FIG. 2K) List of selected genes within ECM, signaling and transcriptional factor (TF) categories and their expression levels in hESC-derived cartilage pellets at days 21 and 45. (FIG. 2L) Relative expression values of hallmark adipogenesis and lipogenesis genes in hESC-derived pellets across days 21, 30 and 45 measured on qRT-PCR. Scale bars: (FIGs. 2C-2F), 2 pm; (FIG. 2B), 10 pm; (FIG. 21), 15 pm; (FIG. 2J), 25 pm; (FIG. 2H), 50 pm; (FIGs. FIG. 2A-2B, 2G), 100 pm.

[0021] FIGs. 3A-3D depict nonlimiting example of a three-dimensional rendering of BODIPY-stained microdissected ear cartilage from one-month-old wild type mouse. FIG. 3A shows a front, top perspective view of the stained ear cartilage. FIG. 3B shows a left side perspective view of the stained ear cartilage. FIG. 3C shows a left plan view of the stained ear cartilage. FIG. 3D shows a rear, bottom perspective view of the stained ear cartilage.

[0022] FIG. 4 depicts nonlimiting example strategies for purification of in vitro grown hESC-derived cartilage organoids. (FIG. 4B) FACS gating strategy for the purification of BODIPY-stained hESC-derived cartilage. Left panel shows strategy for Forward Scatter and Side Scatter gates. Middle panel shows strategy to select single cells using Forward Scatter height vs area gates. Right panel shows two distinct gates for FITC (lipid staining) based on low vs strong intensity.

[0023] FIGs. 5A-5B depict nonlimiting example strategies for purification and in vitro growth of mouse LCs. (FIG. 5A) FACS gating strategy for the purification of BODIPY- stained P13 LCs. Top left panel shows strategy for Forward Scatter and Side Scatter gates. Top middle panel shows strategy to select single cells using Forward Scatter height vs area gates. Top right panel shows two distinct gates for FITC (lipid staining) based on low vs strong intensity. Bottom left panel shows gating strategy to select live cells (negative PI stain). Bottom middle panel shows single cell gating. Bottom right panel shows gating for FITC (lipid staining) following selection of live cells only. (FIG. 5B) FACS-purified LCs can be grown invitro. Top row of FTG. 5B shows BODIPY-stained cell pellets at in vitro day 0. Bottom row of FIG. 5B shows BODIPY-stained cell pellets after 7 days in culture conditions.

[0024] FIGs. 6A-6D depict nonlimiting carton schematics for protocols. (FIG. 6A) Pluripotent cells (embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) are differentiated into a neural crest-like identity (iNCC) in vitro for 3-5 days. iNCCs are then differentiated in vitro using conditioned media into a chondrogenic progenitor (CPC) fate for 21 days. (FIG. 6B) CPCs are incubated in an incubator, or bioreactor for a period of 25-65 days to induce lipid vacuole (LV) formation. LV-containing chondrocytes (LCs) can be identified and separated from lipid-void cells by buoyancy via high speed centrifugation. (FIG. 6C) LCs can be purified by FACS using lipid staining dyes. Purified LCs can then be further grown in incubators or bioreactors to expand cell numbers. New LCs can be purified by FACS as before. (FIG. 6D) Mature LCs can be seeded onto a pre-generated custom-shaped extracellular matrix (ECM) to generate lipocartilage tissues for grafting, based on patient need and specifications.DETAILED DESCRIPTION OF SOME EXAMPLE EMBODIMENTS

[0025] Conventionally, the size, shape, and biomechanics of cartilages are determined by their voluminous ECM. Disclosed herein is the finding that cartilages consist of lipid-filled chondrocytes called LCs. Despite resembling adipocytes, LCs were molecularly distinct and produced lipids exclusively via de novo lipogenesis. Consequently, LCs grew uniform LVs, that resisted systemic lipid surges and did not enlarge upon obesity. LCs also lacked lipid mobilization factors, which enabled exceptional LV stability and protected cartilage from shrinking upon starvation. LVs modulated lipocartilage biomechanics by decreasing the tissue’s stiffness, strength, and resilience. LCs were found in multiple mammals, including in humans.

[0026] While studying mouse ear skin, the inventors noticed prominent “lipid ghost” artifacts in ear cartilage after histological processing. Lipid ghosts are characteristic histological artifacts in adipose tissue, but uncommon in other healthy tissue types where cells lack large LVs. Intrigued by this serendipitous finding, the inventors found that mice possess multiple sites of lipid-laden cartilage (henceforth lipocartilage) consisting of vacuolated, adipocyte-like cells called LCs.Some embodiments of the present disclosure provide methods to:

[0027] (1) Obtain LC progenitors, such as autologous adipose tissue-derived mesenchymal progenitors, autologous mesenchymal stem cells, patient-specific induced pluripotent stem cell (iPSC) derived LC progenitors.

[0028] (2) Differentiate LC progenitors in vitro into differentiated cells (LCs) with head and neck identity and with LVs.

[0029] (3) Purify such differentiated LCs based on their LVs. Purification can be physical, based on buoyance of lipid-filled cells, or can rely on fluorescence-activated cell sorting (FACS) following labeling of LVs with non-toxic small molecule lipid-binding fluorescent dyes.

[0030] (4) Generate new lipocartilage tissue constructs from such purified LCs for use in surgical reconstruction of head (ear, nose) and neck (larynx, trachea) cartilagecontaining body parts, among other applications.

[0031] The present disclosure will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the disclosure in any way.

[0032] Embodiments provided herein are described by way of the following numbered alternatives:1. A method of generating an adult lipocartilage cell (also referred to as LC), the method comprising incubating a progenitor cell capable of differentiating into the adult LC with cartilage-inducing media for at least 20 days.2. The method of embodiment 1, wherein the progenitor cell is a stem cell, optionally wherein the progenitor cell is a pluripotent and / or induced pluripotent stem cell.3. The method of embodiment 1 or 2, wherein the progenitor cell is incubated with cartilage-inducing media for at least 25, 30, 35, 40, 45, 50, 55, 60, or 65 days.4. The method of any one of embodiments 1-3, wherein the progenitor cell is continuously shaken and / or swirled during incubation.5. The method of any one of embodiments 1-4, wherein the cartilage-inducing media is chondrogenic culture and / or differentiation media, optionally wherein the cartilage-inducing media is MesenCult™-ACF Chondrogenic Differentiation Medium for MSCs.6. The method of embodiment 5, wherein the chondrogenic culture and / or differentiation media comprises at least one of: DMEM, high glucose / GlutaMAX, Penicillin, Streptomycin, Fungizone, an at least one amino acid, L-proline, pyruvate, dexamethasone, fetal bovine serum, TGF-bl, bFGF, PDGF, or any combination thereof.7. The method of embodiment 5 or 6, wherein the chondrogenic culture and / or differentiation media comprises at least one of: BMP1, BMP2, BMP5, GDF5, GDF10, IGF1, IGF2, IGFBP2, IGFBP4, IGFBP5, IGFBP6, IGFBP7, TGFB3, DKK3, SFRP1, SFRP2, SFRP5, WIFI, ANGPT4, ANGPTL7, FGF7, FGF18, CYTL1, or any combination thereof.8. The method of any one of embodiments 1-7, wherein the progenitor cell originates from autologous ear cartilage, autologous nasal cartilage, autologous rib cartilage, autologous xiphoid cartilage, autologous articular cartilage, non-cartilage mesenchymal cells, adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, or any combination thereof.9. A method of identifying an adult LC from a population of cells, the method comprising: screening for the presence of a lipid droplet (also referred to as a lipid vacuole, or “LV”) within a cell, wherein the presence of a lipid droplet indicates that the cell is the adult LC.10. A method of purifying an adult LC from a population of cells, the method comprising: i) screening for the presence of a lipid droplet within a cell; and ii) purifying the cell comprising the lipid droplet.11. The method of embodiment 9 or 10, wherein the screening is performed through fluorescent activated cell sorting (FACs).12. The method of embodiment 9 or 10, wherein the screening is performed through a buoyancy assay, wherein the presence of the lipid droplet in the adult LC results in a different cellular buoyancy compared with other cells in the population of cells.13. The method of embodiment 12, wherein the buoyancy assay is centrifugation.14. The method of any one of embodiments 9-13, wherein the presence of the lipid droplet is determined by positive staining from a dye.15. A method of staining an adult LC, the method comprising administering to the adult LC a dye specific for a lipid droplet.16. The method of embodiment 14 or 15, wherein the dye is a fluorescent lipid dye.17. The method of embodiment 16, wherein the dye is a BODIPY dye.18. The method of embodiment 16 or 17, wherein the dye is selected from: BODIPY 493 / 503, BODIPY 500 / 510, BODIPY 530 / 530, BODIPY 558 / 568, BODIPY 576 / 589, BODIPY 581 / 591, LipiDye, LipidGreen, LipidGreen2, LipidTox, SMCy3, SMCy3.5, SMCy5, SMCy5.5, SMCy7, SMCy7.5, or any combination thereof.19. A method of generating a population of adult LCs, the method comprising: i) screening a general population of cells for the presence of a lipid droplet within a cell; ii) purifying the cell comprising the lipid droplet, thus isolating an adult LC; and iii) expanding the adult LC, thus forming the population of adult LCs.20. The method of embodiment 19, wherein the expansion is performed by incubating the adult LC in the same media that the general population of cells was first grown in.21. The method of embodiment 19 or 20, wherein the expansion is performed for up to two weeks.22. The method of any one of embodiments 19-21, wherein the expansion comprises administering a media with insulin, IGF1, BMP1, BMP2, glucose, or any combination thereof.23. The method of embodiment 22, wherein the glucose is present at 5 mM, 7 mM, 10 mM, 15 mM, 25 mM, or any integer that is between 5 and 25 mM.24. The method of any one of embodiments 19-23, wherein the expansion is performed in a bioreactor, spinner flask, roller bottle, multilayered flask, or any combination thereof.25. A composition for use as a synthetic extracellular matrix (ECM) gel, the composition comprising at least one of: Collagen VI, Collagen VII, Collagen VIII, Collagen IX, Collagen X, Collagen XI, Myocilin, Fibronectin, Aggrecan, Tenascin, Thrombospondin, or any combination thereof.26. The composition of embodiment 25, further comprising chitosan.27. A method of generating a replacement tissue construct, the method comprising seeding the population of adult LCs generated from the method of any one of embodiments 19-26 with the composition of embodiment 25 or 26.28. A replacement tissue construct comprising the lipid droplet-containing adult LC generated using the method of any one of embodiments 1-8, or 10-24.29. The replacement tissue construct of embodiment 28 for use in grafting onto a subject.30. The replacement tissue construct of embodiment 28 for use as part of replacement and / or reconstructive surgery in a subject in need thereof.31. The replacement tissue construct of embodiment 29 or 30, wherein the subject is mammalian and / or human.32. The replacement tissue construct of any one of embodiments 29-31, wherein the subject has a head and / or a neck cartilage defect.33. The replacement tissue construct of embodiment 32, wherein the head and / or neck cartilage defect is a defect in nose, ear, laryngeal, or tracheal cartilage, or any combination thereof.34. The replacement tissue construct of any one of embodiments 29-33, wherein the replacement tissue construct provides structural support of non-cartilage craniofacial tissues in the subject.35. The replacement tissue construct of embodiment 34, wherein the non-cartilage craniofacial tissue is skin and / or muscle.36. The replacement tissue construct of any one of embodiments 29-33, wherein the subject requires replacement and / or reconstructive surgery of intervertebral discs.37. A kit for use in tissue grafting, the kit comprising the adult LC generated using the method of any one of embodiments 1-8, or 10-24.38. A method of grafting the adult LC generated using the method of any one of embodiments 1-8, or 10-24 into a subject in need thereof, the method comprising administering the adult LC as part of a suspension.39. A method of grafting the adult LC generated using the method of any one of embodiments 1-8, or 10-24 into a subject in need thereof, the method comprising administering the adult LC cell as part of a cell pellet.EXAMPLES:

[0033] The non-limiting example methodology as disclosed herein are used in the following working Examples.Example 1: Lipocartilage is lipid-filled, long-lived tissue

[0034] Mouse cartilage was comprehensively profiled for the presence of LCs (Fig. 1A). In the head and neck, many cartilage structures arise from the neural crest cells, that express Wnll and label with Wntl-Cre2 genetic construct. In Wntl-Cre2;mT / mG mice, in which JFn / 7-expressing cells and their progenies become stably GFP reporter-positive, the nasal cartilage expressed GFP (n=5), and consisted of vacuolated cells that stained with neutral lipid dye OilRedO (n=3). The larynx, a complex organ in the neck, was comprised of neural crest-derived epiglottic, non-neural crest-derived cricoid and arytenoid, and composite thyroid cartilages (n=5). With exception of the non-neural crest portion of the thyroid cartilage, the inventors found lipid-laden cells in all laryngeal cartilage elements analyzed (n=3). Lipid-filled cells also constituted the xiphoid cartilage of the sternum (n=5). Ear cartilage stained strongly with OilRedO (n=8) and the fluorescent neutral lipid dye BODIPY (n=3). Its lipid-filled cells were highly uniform in size (Fig. IB), and organized into a thin plate, two-to-three cell diameters across. In many places, the ear cartilage plate had fenestrae commonly traversed by small blood vessels and occupied by clusters of large lipid-filled cells. In Wntl-Cre;R26R mice (n=4) and cartilage-specific Col2al -CreERT;R26R mice (n=3), in which Cre-expressing cells stably activate P-Galactosidase and label with lacZ dye, only lipid-laden cells in the plate, but not those in fenestrae, were lacZ-positive. Conversely, in adipocyte-specific Retn-lacZ (n=4) and Adipoq-Cre;R26R mice (n=6) only lipid-filled cells of fenestrae expressed lacZ. Thus, LCs are prevalent across numerous cartilages and have a distinct embryonic origin than similar-looking bona fide adipocytes.

[0035] The inventors then examined LCs’ longevity. Low dose tamoxifen treatment of Col2al-CreERT;R26R mice at postnatal day P6 recombined individual lipocartilage progenitors, leading to discretely labeled LC clones in adult ears. LC clone sizes 3 weeks (n=449 clones) and 23 weeks after labeling (n=384 clones) were not significantly different (p=0.506). Furthermore, large BODIPY+LCs persisted in the nose of aged mice (n=4) and ears of aged rats (n=6), supporting the notion that LCs are long-lived cells with a limited turnover rate.Example 2: Lipid droplets modulate tissue biomechanics

[0036] Next, the inventors performed a series of uniaxial tensile tests to compare biomechanical properties of one-month-old ear lipocartilage (n=6), inguinal white adipose tissue (WAT; n=9), knee meniscus (n=7), and rib cartilage (n=8). Young’s modulus and ultimate tensile strength values of lipocartilage (3.25±0.38 and 1.30±0.24 MPa, respectively) were significantly higher than those of WAT (0.11±0.04 MPa (p=0.0004) and 0.11±0.02 MPa (p=0.005), respectively) (Fig. IB), indicating a greater ability of lipocartilage to resist deformation and tear. Lipocartilage also showed significantly lower strain at failure values (0.60±0.1) compared to adipose (1.61±0.19; p=0.0008) (Fig. IB). In contrast, ECM-rich and lipid-devoid cartilage from knee meniscus and rib showed significantly higher Young’s modulus (23.94±5.14 and 23.86±4.32 MPa, respectively) and ultimate tensile strength values (9.86±2.61 and 11.01=1=1.91 MPa, respectively) that fell within previously reported values for fibrous and hyaline cartilages. The inventors then asked whether removal of lipid vacuoles would significantly alter lipocartilage biomechanics. Chloroform / methanol treatment efficiently emptied lipid droplets from LCs, and delipifies microdissected rat ear lipocartilages. Delipified lipocartilages (n=8) showed altered biomechanical parameters compared to their native state (n=8) — Young’s modulus, ultimate tensile strength, and resilience values significantly increased (3.27±0.52 vs. 5.79±0.99 MPa (p=0.0004); 1.91±0.22 vs. 4.53±0.92 MPa (p=0.0259); and 0.38±0.06 vs. 1.77±0.48 MJm'3(p=0.0228); respectively) (Fig. 1C), suggesting that lipid vacuoles conferred compliance to cartilage. Intriguingly, in their delipified state, lipocartilage properties shifted toward values displayed by naturally lipiddevoid and ECM-rich murine meniscus and rib cartilages . Thus, lipid vacuoles impart distinct biomechanical properties to lipocartilages.Example 3: Lipocartilage has unique molecular properties

[0037] Next, the inventors asked what molecular features characterize lipocartilage and how they may differ from those in WAT. The inventors started by comparing whole-tissue lipidomic profiles of one-month-old ear lipocartilage (n=5) and inguinal WAT (n=5). Upon analysis, neutral lipids appeared as the dominant molecular species of both tissues owing to the large size and abundance of their lipid vacuoles. With this in mind, the inventors found that the composition of lipocartilage vacuoles differs from that of WAT. In WAT, 86.5% of neutral lipids were triglycerides. However, in lipocartilage triglycerides constituted only 43.3% ofneutral lipids, and 31.1% (three times higher than in WAT) were fatty-acid-esters-of-hydroxy- fatty-acids (FAHFAs) — a class of neutral lipids with anti-inflammatory properties. Compositional lipidome differences between lipocartilage and WAT extended across all classes of lipids beyond triglycerides and FAHFAs. Furthermore, compared to WAT (3.8%), lipocartilage lipids also contained significantly more saturated fatty acid chains (19.7%), the main product of de novo lipogenesis. Thus, lipid vacuoles in lipocartilage are produced using metabolic pathways distinct from those in WAT.

[0038] The inventors then compared ear lipocartilage (n=3) and WAT (n=3) using whole-tissue mass spectrometry. In both tissues, the inventors separately studied ECM proteins, which dominate upon analysis, as well as those involved in lipid metabolism. Lipocartilage and WAT displayed significant compositional differences in both protein categories. WAT ECM was dominated by collagen I and III subunits (COL1 Al, 1 A2 and 3A1), which collectively accounted for 97% of all detected ECM proteins. In contrast, these three collagens constituted only 62.2% of ECM proteins in lipocartilage, which also featured signature cartilage proteins COL2A1, 6A1, 6A2 and elastin, as well as ECM proteins normally not associated with cartilage: myocilin (8.4%) and collagen COL8A1 (2%). Upon immunostaining, COL8A1 dominantly localized to the lipocartilage plate periphery, while myocilin surrounded individual mature LCs. In the lipid metabolism category, the topmost abundant factors in WAT were de novo lipogenesis enzyme fatty acid synthase (FASN, 15.5%), fatty acid binding protein FABP4 (14.3%), lipid mobilization proteins PLIN4 (9.8%) and PLIN1 (6.6%), and fatty acid transporter CD36 (8.2%). Of these, only FASN (5.4%) was significantly detectable in lipocartilage by immunostaining (Fig. 2H, 21), whose other topmost lipid metabolism factors were apolipoprotein E (APOE, 14.5%) and PLIN2 (2.4%). Notably, the third most abundant protein in lipocartilage was vimentin, known for forming intermediate filament cages around early lipid vacuoles. Vimentin was 15 times more abundant in lipocartilage compared to WAT. Thus, despite sharing a similar morphology, both tissues have distinct ECM composition and significant differences in lipid biogenesis machinery.

[0039] Next, the inventors compared adult isolated ear LCs (n=3), WAT adipocytes (n=3) and rib hyaline cartilage chondrocytes (n=3) by RNA-sequencing. The LC transcriptome was distinct from those of adipocytes and hyaline chondrocytes with 8,455 differentially expressed genes. Each cell type was enriched for unique Gene Ontology terms with LCssimultaneously enriched for cartilage and metabolic terms. Both LCs and hyaline chondrocytes expressed many of the same ECM genes that were otherwise minimally present in adipocytes, yet also showed significant cartilage type-specific enrichment patterns. Matching the proteomic data, LCs expressed particularly high levels of myocilin and Col8al. Significant expression differences between LCs and hyaline chondrocytes extended across signaling molecules and transcriptional regulators . Matching and expanding on the proteomic results, LC and adipocyte transcriptomes prominently diverged within the metabolic category. Unlike adipocytes, LCs did not express adipokines Adipoq, Cfd (adipsin), Lep, Retn, Retnla and pro- adipogenic transcriptional regulators Ebfl, Ebf2, Cebpb, Ppara, Rxrg, Thrsp. Differences extended to multiple key genes involved in lipid biosynthesis and catabolism, as well as lipid vacuole formation and maintenance. LCs did not express Cd36 and express low levels of Slc27af two major fatty acid transporters in adipocytes. However, glucose transporters, including Slc2al and Slc2a4. and key de novo lipogenesis enzymes Acly, Acaca, Fasn, and Scdl were expressed. LCs also expressed many key enzymes involved in triglyceride synthesis. Notably, Pnpla2, Lipe, and Mgll, which are each important enzymes in triglyceride mobilization and breakdown were expressed at very low levels in LCs. Also, not expressed and downregulated, respectively, were Plinl and Abhd5, both of which are critical for hormone-stimulated lipolysis in adipocytes. Finally, LCs expressed several key proteins implicated in lipid vacuole assembly, fusion and stability, albeit many of them at reduced levels as compared to adipocytes. The inventors validated selected differentially expressed metabolic genes by qRT-PCR and by in situ RNA staining.

[0040] Thus, the combined multiomic profding disclosed herein reveals that lipocartilage is a distinct skeletal tissue type, whose ECM only partially resembles conventional cartilage ECM and whose lipid-filled cells lack many important fat biogenesis genes, otherwise expressed by metabolically active adipocytes.Example 4: Lipocartilage differentiates via lipogenesis

[0041] It was next asked when during their development lipocartilage activates the lipogenesis program. Distinct forms of lipocartilage in mice develop asynchronously — for example, while nasal LC formed lipid droplets during embryonic development, starting from E13.5, ear LC did so with a significant delay, starting around postnatal day P10. Given its accessibility and ease of microdissection, the inventors profiled ear lipocartilage developmentin-depth. By examining morphological changes, co-expression patterns of chondrogenic marker SOX9 and proliferation marker PCNA as well as SOX9 and EdU incorporation (Fig. Si l), and lipid vacuole marker OilRedO, the inventors established the following key morphogenetic events: P4 as the onset of chondrogenic commitment by ear pinna mesenchyme; P8 and PIO as the early and late proliferative phases, respectively; Pl 3 as the transition phase; and P21 and one month as the early and late differentiation phases, respectively. The inventors then studied LCs and their progenitors at five time points by RNA- sequencing. Between P8-P13, the inventors sorted LC progenitors as tdTomato111cells from micro-dissected Wntl-Cre2;tdTomato cartilage primordia. At P21 and one month, lipid- containing LCs were isolated by buoyancy. The LC transcriptome prominently changed with time (n=3 / time point) and 2,397 differentially expressed genes organized into four timedependent clusters. Cluster T1 genes upregulated during the proliferative phase; T2 genes — during the transition phase; T3 and T4 genes — during early and late differentiation phases, respectively. Gene category and Gene Ontology enrichment analyses showed that cell cycle activators and pro-mitotic factors were abundant in LC progenitors, and then largely shut down upon differentiation. In contrast, cell cycle inhibitors were enriched in late differentiated LCs. Lipogenesis genes, including Dgat2, Fasn, Lpinl, and Mogatl prominently increased at the onset of LC differentiation, while other critical lipogenesis factors, including Agpat2, Agpat3, Bscl2, Cidea, Dgatl, Fabp5, Mogat2, and Plin2 were enriched in late differentiated LCs. Transcription factors (Fig. 3D, jade) and ECM genes (Fig. 3D, purple) showed early and late enrichment peaks. Early transcription factors included neural crest-derived mesenchyme regulator Hesl and chondrogenesis regulators Sox6 and Sox8. Late transcription factors included known lipogenesis activators Klfl5, Srebfl, Stat5a and Stat5b. Early ECM genes included established cartilage-specific collagens Col2al, 9al, 9a2, lla2, as well as elastin, fibulin 5, fibrilin 2 and periostin, while late ECM genes included collagens Col8al, CollOal, as well as chondroadherin, fibrilin 1 and myocilin. Signaling factors showed complex temporal patterns across several major pathways, including BMP, IGF, TGFp, WNT. Thus, lipocartilage developmental program is dominated by cell cycle genes, pro-chondrogenic transcription and ECM factors during its early phase, and by lipogenesis machinery and specialized ECM proteins in the late phase.

[0042] Because the growth of lipid vacuoles in LCs resulted in a form of cellular hypertrophy, the inventors asked if lipocartilage activates canonical cartilage hypertrophy genes and form templates for mineralizing bones. Unlike rib chondrocytes, LCs did not express cartilage hypertrophy markers Alpl, Ihh and Rurix2 on RNA-seq. Staining of embryonic nasal capsule for BODIPY and ossification marker collagen COLX in E13.5-E15.5 mice and spatial sequencing analysis in E16.5 mice showed that differentiating nasal lipocartilage was sharply demarcated from nasal septum bone and did not activate the ossification gene program. Thus, lipocartilage does not ossify and LC differentiation is distinct from canonical chondrocyte hypertrophy via cytoplasmic swelling.Example 5: Lipocartilage cells are metabolically refractory

[0043] LCs did not express Cd36 and prominently downregulated Slc27al, both necessary for dietary fatty acid uptake in adipocytes. Furthermore, genes necessary for fatty acid mobilization from lipid stores were either not expressed — Plinl, or strongly downregulated — Abhd5, Lipe, Mgll and Pnpla2. This prompted the inventors to test how LCs respond to fluctuations in dietary lipid availability. The inventors studied ear morphology and LC size in mice after twelve weeks of high fat diet or 72 hours of restricted caloric intake. Compared to mice fed regular chow (n=6), ear size ) and lipid droplet size did not change significantly in mice fed a high fat diet (n=6; p >0.9999 and p>0.1966, respectively). This was despite significant increases in body weight (p=0.0008) and dermal and perigonadal WAT adiposity. Similarly, obese leptin receptor null mice (db / db; n=6) showed a significant increase in body weight (p>0.0005) and WAT adiposity, but no significant changes in ear size (p=0.1733) or LC size (p=0.9261). Mice on restricted caloric intake (n=3) also did not show significant ear or LC size changes (p >0.9999 and p>0.6033, respectively), even though their body weight (p=0.0277) and WAT adiposity reduced significantly. Thus, unlike WAT, lipocartilage is protected from systemic fluctuations in dietary lipid availability.

[0044] To directly examine dietary lipid uptake, the inventors injected mice with the fluorescently labeled fatty acid C1-BODIPY-C12. Six hours after a single injection in one- month-old mice (n=3), C1-BODIPY-C12 substantially incorporated into perigonadal WAT but was undetectable in ear lipocartilage. The inventors also performed daily probe injections for 8 days starting at P10 (n=3) and observed C1-BODIPY-C12 broadly incorporated into perigonadal WAT as well as into adipocyte clusters within ear cartilage fenestrae, but not intoLCs. To examine lipid catabolism, the inventors compared one-month-old lipocartilage and WAT by fluorescence lifetime imaging microscopy (FLIM) at 740 nm excitation wavelength. This approach identifies endogenously fluorescent, long lifetime molecular species (LLS) that serve as a label-free marker of lipid catabolism and peroxidation. The inventors saw a strong LLS signature that, when mapped back to the original images, localized to lipid vacuoles in perigonadal adipocytes (n=4) and adipocytes within ear cartilage fenestrae (n=5), but not in LCs (n=5). Thus, in contrast to adipocytes, LCs have stable lipid vacuoles that are insensitive to fluctuations in dietary fat and are shielded from lipid catabolism. The gene expression signature of LCs, depleted of key fatty acid transporters and neutral lipid mobilization factors, was consistent with the observed biology.Example 6: Lipocartilage cells are typically a product of de novo lipogenesis

[0045] Lack of dietary lipid uptake in LCs suggests that their vacuoles must rely on endogenous production of lipids from glucose via de novo lipogenesis. To directly test this assumption, the inventors imaged the incorporation of glucose-derived deuterium into lipid vacuoles of developing LCs using stimulated Raman scattering (SRS) microscopy. This approach detects vibrational properties of molecules, such as carbon-hydrogen (C-H) bonds in lipids. The inventors cultured P14 mouse ears for 72 hours in high glucose control media (n=3) or in glucose-free media supplemented with deuterated glucose (G-d7) (n=3). A strong carbondeuterium (C-D) signal was detected in lipid vacuoles of G-d7-exposed LCs but not control LCs. In the Raman spectrum, the C-D signal observed in G-d7-exposed LCs matched the C-D signal profile of pure G-d7 and spatially overlapped with the strong C-H signal of LC lipid vacuoles, indicating incorporation of deuterium into LC lipid stores. Considering that normal fasting blood glucose levels in mice are 80-100 mg / dL (equivalent to 4.4-5.5 mM), the inventors also cultured microdissected P16 mouse ear lipocartilage in media containing either low (4 mM) or high (25 mM) concentration of glucose. Upon BODIPY staining after 5 days, cultured lipocartilage showed significantly larger lipid droplets relative to culture day 0 (p<0.0001), with lipocartilage cultured under high glucose (n=4) also showing significantly larger lipid droplets relative low glucose-exposed lipocartilage (n=4) (p<0.0001).

[0046] Next, the inventors disrupted de novo lipogenesis pathway in vivo. Starting at P10, the inventors performed daily topical applications of AcetyLCoA carboxylase inhibitors PF-05175157 and ND-646 or with C75, a fatty acid synthase inhibitor, on mouseears. By P21, in all three cases, the inventors observed reduction in ear sizes, with ND-646 and C75 inducing prominently misshapen ears, 88±2% and 55±7% smaller in size, respectively, compared to DMSO-treated controls. Upon BODIPY staining, parts of ear cartilage in C75- treated mice contained large areas devoid of lipid vacuoles. Thus, differentiating LCs grow lipid vacuoles via de novo lipogenesis from glucose.Example 7: Lipocartilage cells form in human cartilage

[0047] Next, the inventors examined human fetal cartilage at gestational week (GW) 20-21. At this stage, ear cartilage had undergone maturation and was capable of strongly staining with sulfated glycosaminoglycan dye Alcian blue (FIG. 2A) and expressed collagen COL8A1. Remarkably, this cartilage also contained numerous lipid vacuoles (FIG. 2B), despite not expressing adipocyte-specific marker PLIN1. Frequent and large lipid vacuoles were confirmed in ear, nasal, as well as thyroid and epiglottic GW20-21 human cartilage by transmission electron microscopy (FIG. 2C-2F). Next, the inventors studied cartilage organoids generated in vitro from a human embryonic stem cells (hESCs)-derived neural crest intermediate. Similar to fetal ear cartilage, day 65 cartilage organoids strongly stained with Alcian blue (FIG. 2G) and contained numerous OilRedO+lipid vacuoles (FIGs. 2H, 21). Lipid content of these vacuoles and the presence of a collagen-rich ECM was confirmed by SRS microscopy and second-harmonic generation, respectively (FIG. 2J).

[0048] To gain a molecular insight into human cartilage lipogenesis, the inventors profiled hESC-derived cartilage organoids on RNA-sequencing at days 21 (n=3), 30 (n=3) and 45 of differentiation (n=4). Day 21 was chosen as the starting point, because organoids first become Alcian blue-positive at this stage. On analysis, organoids prominently segregated by time point, with 4,715 differentially expressed genes. Many of these genes paralleled the expression dynamics observed during mouse lipocartilage development, including within ECM, signaling, transcription factor and lipid biogenesis categories (FIG. 2K). Notably, analogous to mouse LCs, human organoids expressed lipogenic factor SREBF1 but not PPARG. They also lacked expression of adipokines^ZYPOO and RETN, or CD36 and PLIN1, which are important for fatty acid uptake and mobilization, respectively. At the same time, like mouse LCs, they prominently expressed core de novo lipogenesis factors ACLY, ACACA, FASN, SCD; triglyceride synthesis factors, including GPAT4, AGPAT1, DGAT1; and lipid droplet biosynthesis genes, including CAV1, LPL, FABP3 and PLIN2 (FIGs. 2K, 2L). Thus,lipid vacuole formation is an integral part of human craniofacial cartilage differentiation — a program that is recapitulated in hESC-derived cartilage organoids.Example 8: Lipocartilage is prevalent in mammals

[0049] Next, the inventors examined if lipocartilage exists in mammals from distinct phylogenetic groups. Owing to the ease of collection from preserved museum samples, the inventors primarily focused our study on the external ear, a distinguishing feature of most extant mammals, with its earliest paleontological evidence dating to Early Cretaceous, 125 million years ago. The inventors examined ear cartilage from a total of 65 species, covering 4 orders of marsupials and 18 orders of eutherians, and found lipocartilage in multiple species across the clade. The inventors observed that prominent lipid-filled cells existed in the ear cartilage of species with thin, membrane-like ears, irrespective of their phylogeny. However, ear lipocartilage morphology was diverse across species. In rodent species, such as spiny mice (Acomys cahirinus') or jerboa (Jaculus jaculus) , lipocartilage generally resembled that of Mus musculus - a flat plate of tightly-packed LCs with fenestrae, some of which contain adipocyte clusters. Notably, the inventors also saw fenestrated lipocartilage in marsupial species, where, in addition to adipocytes, fenestrae could contain pigmented material, as seen in kowari (Dasyuroides byrnei) or opossum (Didelphis virginiana). In other marsupials, such as the squirrel glider (Petaurus norfolcensis), many of the fenestrations nestled hair follicles, one per opening. In bat species, such as in Pallas’s long-tongued bat (Glossophaga soricina), LCs differentially distributed across lipocartilage, sparsely along the medial side and in shape of periodic parallel ridges along the lateral side. Ridges consisted of “stacks” of lipid-laden cells and gave the ear a “ruffled” morphology. Other bat and rodent species also showed clear LCs in nasal cartilage. Prominent lipid vacuoles were also found in ear cartilage of some Afrotherians, such as the eastern rock elephant shrew (Elephantulus myurus).

[0050] Finally, the inventors inquired about the presence of lipocartilage in nonmammalian tetrapods. The inventors performed Alcian Blue and OilRedO staining on tracheal and peri -tracheal cartilages from two amphibians — Forrer’s grass frog (Lithobates forreri) and axolotl (Ambystoma mexicanump chicken (Gallus gallus) and three reptilians — American alligator (Alligator mississippiensis), green anole (Anolis carolinensis) and red-eared slider turtle (Trachemys scripta elegans). In all instances, cartilage did not contain lipid droplets and, instead, had the morphology of canonical ECM-rich hyaline cartilage. The inventors thus positthat lipid-filled cells are a common feature of cartilage across the mammalian clade, and exclusive among other tetrapods.Example 9: Discussion

[0051] As disclosed herein, it was determined that LCs are not adipocytes, but instead a type of lipid-filled skeletal cell. A unique property of LCs is their ability to form super-stable lipid vacuoles. Unlike adipocytes, LCs maintain stable lipid vacuoles upon maximal obesity as well as acute starvation. LCs manage to “untether” from systemic metabolism by not expressing fatty acid transporters and other key lipid-mobilization factors. Longevity of LCs and their ability to tolerate high content of otherwise cytotoxic lipids present an opportunity to use them in studying mechanisms of cellular aging. In addition, the uniformity and extreme stability of LCs’ lipid vacuoles hold new clues to mechanism of cell size control.

[0052] Enlargement of LCs via lipid droplets is a form of cellular hypertrophy. Differentiating chondrocytes in hyaline cartilage poised for ossification also undergo hypertrophy via cytoplasmic swelling. However, molecular aspects of the hypertrophy program in LCs are distinct from those in hyaline chondrocytes, and unlike hyaline cartilage, lipocartilage is not a template for ossification, but rather a permanent lipid-filled skeletal structure. Studying how lipid-filled LCs maintain a “healthy” cell state relative to diseased lipid-filled articular chondrocytes can offer new molecular interventions strategies for cartilage diseases.

[0053] Disclosed herein are some structure-function relationships in skeletal tissues. Key mechanical properties of cartilage — stiffness and strength, have been attributed to their ECM composition and organization. The present findings indicate that lipocartilage displays robust mechanical properties despite consisting of giant lipid-filled cells with minimal ECM. Currently, cartilage biomechanics are commonly described using continuum theory, assuming that ECM is homogeneous and that mechanical contribution from cells is negligible.

[0054] Lipocartilage in many species have an intricate morphology, with those in bat ears being particularly complex, featuring arrays of parallel and interconnected microridges, thought to be involved in proper sound perception. Here, it is reported that such microridges result from the precise stacking of individual LCs.

[0055] The inventors propose that lipocartilage is an outcome of convergent evolution and is analogous to the notochord. The latter is an ancient skeletal tissue type that first appeared in early Chordates, and that in extant mammals forms the nucleus pulposus of the inter-vertebral discs. One morphological feature of the notochord and its derivatives is their giant, tightly packed vacuolated cells. However, unlike the LCs in lipocartilage, cells of the notochord contain aqueous rather than lipid vacuoles. Biomechanical properties of the notochord are determined by virtue of hydro-static forces generated between its vacuolated cells. As supported by delipification experiments, biomechanical properties of lipocartilage arise, at least in part, from the “lipo-elasticity” of LCs.

[0056] The inventors also propose that the high and molecularly distinct lipid content of ear lipocartilage may aid in the ear pinna's ability to gather and focus acoustic waves. Indeed, animals are shown to utilize lipid-rich tissues in bioacoustics.

[0057] Abundant lipid vacuoles form in neural crest-derived cartilage organoids induced in vitro from human ESCs. The inventors propose that lipid vacuoles can be used as a biomarker for purification of differentiated human cartilage cells in ESC-derived cultures. A major hurdle for using ESC-derived cells in regenerative medicine is the inability to reliably separate differentiated cells from pluripotent progenitors, which present a risk for teratoma development. Lipid vacuoles can be labeled easily and reliably with intra-vital fluorescent lipid dyes and labeled differentiated LCs can be purified by FACS. Application of lipid vacuolebased cell purification to ESC- or iPSC-derived lipocartilage organoids could potentially help advance regenerative approaches to cartilage repairs.

[0058] In this study, the inventors characterized a distinct type of lipid-rich skeletal tissue present in many mammals. This tissue develops from embryonic progenitors that first acquire a chondrogenic cell fate, but then proceed to differentiate by synthesizing giant lipid vacuoles — a distinct mechanism from conventional cartilage. The inventors showed that the resulting lipid vacuoles are super-stable and do not contribute to systemic lipid metabolism but, instead, confer the tissue with its biomechanical properties. Our observations lay a foundation for studies on the non-metabolic roles of lipid vacuoles in skeletal tissue morphogenesis, homeostasis and function.Example 10: Materials and methods

[0059] Experimental mouse models. Wntl-Cre2 (JAX, #022137), rnTinG (JAX, #007576), R26R (JAX, # 003309), tdTomato (JAX, #007914), Col2al-CreERT(JAX, #006774), Retn-lacZ, Adipoq-Cre (JAX, #010803), db / db (JAX, #000697), and C575Z / 6JDIO (JAX, # 380050) were used. In Col2al-CreERT;R26R mice, tamoxifen in corn oil was injected IP for four consecutive days starting at P21 at a dose of 75 mg / Kg for full induction. For low- dose induction, a single dose of tamoxifen in com oil at 7.5 mg / Kg was injected IP at P6.

[0060] Lipidomic and proteomic profding. All lipid standards were acquired from Cayman Chemical Company, Matreya, Cambridge Isotope Laboratories, NuChek Prep, Avanti Polar Lipid, or Sigma-Aldrich. All solvents were of high-performance liquid chromatography (HPLC) or liquid chromatography -mass spectrometry (LC / MS) grade and were acquired from Sigma-Aldrich, Fisher Scientific, or VWR. Medial, fenestrae-free zones of the ear cartilage plate were dissected from adult P33 C57B1 / 6J female mice (at least 10 mg per sample), carefully avoiding adipocytes. For white adipose tissue control, inguinal fat was collected (at least 10 mg per sample). All tissues were rinsed thoroughly in IX PBS and flash frozen for downstream use.

[0061] For lipidomic analysis, tissues were thawed in ten times diluted PBS and homogenized in Omni bead tubes with 2.8-mm ceramic beads in the Omni Bead Ruptor 24 with Cryo Cooling Unit (Omni International) at 4 °C for 2 minutes. Protein concentration was determined by the bicinchoninic acid assay. 1 mg of protein from each sample was aliquoted, and a cocktail of deuterium-labeled and odd chain phospholipid standards from diverse lipid classes was added. Standards were chosen so that they represented each lipid class and were at designated concentrations chosen to provide the most accurate quantitation and dynamic range for each lipid species. 4 mL chlorofornrmethanol (1 : 1, by volume) was added to each sample, and lipidomic extractions were performed. Lipid extraction was automated using a customized sequence on a Hamilton Robotics STARlet system (Hamilton). Lipid extracts were dried under nitrogen and reconstituted in chloroform :methanol (1 : 1, by volume). Samples were flushed with nitrogen.

[0062] Samples were diluted 50 times in isopropanol:methanol:acetonitrile:water (3:3:3: 1, by volume) with 2 mM ammonium acetate in order to optimize ionization efficiency in positive and negative modes. Electrospray ionization-MS was performed on a TripleTOF5600+ (SCIEX), coupled to a customized direct injection loop on an Ekspert microLC200 system (SCIEX). 50 mL of sample was injected at a flow rate of 6 mL / min. Lipids were analyzed using a customized data independent analysis strategy on the TripleTOF 5600+ allowing for MS / MSALL high-resolution and high-mass-accuracy analysis. Quantification was performed using an in-house library on MultiQuant software (SCIEX) and normalized to 1 mg protein.

[0063] For proteomic analysis, tissues were digested in mass spectrometry grade trypsin at 65°C overnight to form a complete, homogeneous peptide digest. Digests were desalted using C18 cartridges (Waters Sep-Pak) per manufacturer’s protocol, concentrated under vacuum, and resuspended in 0.1% formic acid. Peptides underwent bottom-up proteomics analysis with label-free quantification (LFQ). Briefly, samples were injected into a Thermo Fischer Scientific UltiMate 3000 RSLC system coupled to a Thermo Fischer Scientific Orbitrap Fusion Lumos mass spectrometer at a flow rate of 300 nl / min. Identification and LFQ were performed using MaxQuant software. Resulting LFQ values were used for comparing the relative abundance of proteins among different samples, normalized to total protein content.

[0064] Tissue delipification for mechanical testing. Adult rat ear cartilage tissue was dissected and rinsed in fresh IX PBS. Using a tissue biopsy punch, 6 mm diameter discs were prepared and either left intact as native tissue controls or processed through a methanol gradient in one-hour 25% increments until absolute methanol. Samples were then incubated twice in fresh absolute methanol for a minimum of 15 minutes, then transferred into a 2: 1 chloroform-methanol solution for three hours in a rocking platform, then taken through a reverse methanol gradient into IX PBS and rinsed at least three times before downstream analysis.

[0065] Mechanical testing and analysis. For mechanical testing, uniaxial tensile tests were performed. Briefly, tissues are dissected and rinsed in cold IX PBS. Note that for delipification studies, control and delipified tissues were generated as described in the previous subsection. Tissues are then cut into bone-shaped pieces and imaged to obtain dimensions via ImageJ analysis. Samples were paper tabs, loaded into an Instron Model 5565, and pulled to failure at a rate of 1% strain per second. Force-displacement curves were used to calculatetensile Young’s modulus, ultimate tensile strength (UTS), and ultimate tensile strain using a custom MathWorks’ MATLAB code.

[0066] Histology and immunostaining. For hematoxylin and eosin (H&E) staining, PFA-fixed sections were immersed in Harris’ hematoxylin and eosin solutions (National Diagnostics) following the manufacturer’s suggestions, dehydrated, cleared and mounted in toluene. For lacZ staining, tissues were fixed in 4% PFA for 30 minutes at room temperature, rinsed in IX PBS and incubated in lacZ buffer at 37 °C for 5 minutes, then transferred to lacZ buffer with X-Gal (VWR) and incubated at 37 °C until signal developed. For OilRedO (VWR AMRESCO) staining, a 6.25 mg / ml stock solution in isopropanol was prepared and diluted as a working solution to 60% in H2O followed by filtration immediately before staining. Tissues were fixed in 4% PFA for 8 hours at 4 °C, rinsed twice in H2O, then submerged in 60% isopropanol for 1 minute followed by staining in OilRedO working solution for 30 minutes. Tissues were then rinsed twice in 60% isopropanol followed by H2O and imaged in IX PBS. For BODIPY staining, tissues were incubated in 200 pmolar BODIPY 493 / 503 (Thermo Fisher) in IX PBS for 30 minutes, rinsed twice in IX PBS and imaged. For Alcian blue staining, fixed tissue sections were immersed in 1% Alcian blue 8GX in 3% aqueous acetic acid (pH 2.5) for 30 minutes, rinsed in distilled water, and, for fetal ear, counterstained with Harris hematoxylin before procedural dehydration and mounting. The primary antibodies used were rabbit anti-COL8Al (1 :500; Antibodies-Online), rat anti-CD31 (1 :250; Invitrogen), mouse anti-MYOC (1 :250; ProteinTech), rabbit anti-FASN (1 :250; Cell Signaling Technology), rabbit anti-PLINl (1 :750; Cell Signaling Technology), rabbit anti-SOX9 (1 :250; Millipore), and mouse anti-PCNA (1 :200; Abeam). Secondary antibodies used were Alexa Fluor 488 and 555 goat anti-rabbit (ThermoFisher) and goat anti-mouse (ThermoFisher) at 1 : 1000. Stained sections were mounted with non-hardening mounting medium with DAPI (Vector Labs). For mouse anti-PCNA and rabbit anti-SOX9, immunostaining was performed on paraffin sections with heat-based antigen retrieval; for other antibodies frozen tissue sections were used.

[0067] RNAscope in situ assay. Mouse ear pinna tissue sections were used for RNA in situ hybridization using RNAscope® kit v2 (323100, Advanced Cell Diagnostics), following the manufacturer’s instructions. The following Mus musculus probes from Advanced Cell Diagnostics were used: Mm-Col8al (51807), Mm-Fasn-C2 (490661-C2), Mm-Acaca-C2(576581-C2), Mm-Dgat2-C2 (481301-C2), Mm-Cd36-C2 (464431-C2), Mm-Acly-C2 (460391-C2), Mm-Sox9-C3 (401051-C3).

[0068] RNA extraction and RNA-sequencing. Mouse ear cartilage tissues were collected from Wntl-Cre2;tdTomato mice at several postnatal time points. In P8, PIO, and P13 mice, red fluorescence-positive ear cartilage was microdissected and incubated in 0.75 mg / ml Collagenase I (Sigma- Aldrich) in 10% FBS DMEM for 3 hours at 37 °C in 5% CO2 with constant motion. The cell suspension was passed through a 40 pm nylon cell strainer (Corning) and centrifuged at 1,200 RPM. The cell pellet was reconstituted in IX PBS and stained with Zombie Violet Cell Viability Kit (BioLegend) according to the manufacturer’s protocol. Single, live cell fractions were then FACS-sorted as tdTomato111using a BD FACSAria II flow cytometer (BD Biosciences). In P21 and P33 mice, the ear cartilage plate was microdissected and the medial and distal portions of the plate were separated and incubated in Collagenase I. Dissected inguinal adipose tissue was also treated to isolate single adipocytes. Cells were passed through a 100 pm nylon cell strainer (Corning) and centrifuged at 1,200 RPM. Following centrifugation, the buoyant, lipid-filled cell fraction was collected. Sorted tdTomatohlcells (from P8, P10, and P13) and buoyant cells (from P21 and P33) were re-suspended in RLT buffer (QIAGEN) with 1% beta-mercaptoethanol and homogenized. Similarly, hESC-derived cartilage pellets were collected at Differentiation days 21, 30, and 45, rinsed three times in IX sterile PBS, and homogenized in RLT buffer with 1% beta- mercaptoethanol. Total RNA was isolated using the RNEasy Micro-Kit (Qiagen). Optimal- quality RNAs with RIN scores more than 8.8 were considered for cDNA library preparation. Full-length cDNA library amplification was performed by: 1 ng of total RNA was reversed- transcribed, and resulting cDNA was pre-amplified for 13 cycles. Tagmentation was carried out on 18 ng cDNA using the Nextera DNA Sample Preparation Kit (Illumina) at 55 °C for 5 minutes and purified using PCR Purification Kit (Qiagen). Transposed cDNA was used for limited cycle enrichment PCR using previously published Nextera PCR primers. Libraries were amplified for 7 continuous cycles and purified with AMPure XP beads (Beckman Coulter). Library quantification was done using KAPA for Illumina Sequencing Platforms (Illumina). Libraries were multiplexed and sequenced as paired-end on an Illumina Next- Seq500 platform (Cluster density = 296K / mm2, Clusters PF = 71.2%, Q30 = 87.6%). Thelibraries were sequenced to an average depth of 10-30 million reads per library using paired 43bp reads.

[0069] Gene expression analysis. Reads were first aligned using STAR v.2.4.2a with parameters outFilterMismatchNmax 10 — outFilterMismatchNoverReadLmax 0.07 — outFilterMultimapNmax 10’ to the reference mouse genome (mml0 / genocode,vM8), or, in the case of hESC-derived cartilage pellets, aligned to the reference human genome (GRCh38). Gene expression levels were quantified using RSEM v.1.2.25 with expression values normalized into Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Samples with >1,000,000 uniquely mapped reads and >60% uniquely mapping efficiency were used for downstream analyses. Differential expression analysis was performed using edgeR v.3.2.2 on protein-coding genes and IncRNAs. Differentially expressed genes were selected by using fold change (FC)>2, false discovery rate (FDR)<0.05 and counts per million reads (CPM)>2.

[0070] On analysis, numerous genes encoding secreted signals and transcriptional regulators were differentially expressed between LCs and rib chondrocytes. In terms of secreted signals, both cartilage cell types expressed BMP ligands, but LCs were enriched for Bmp5, while rib chondrocytes for Bmp4 / 6 7'8a. Among TGFp ligands, LCs express Gdf5 and pfb3, while rib chondrocytes expressed GdflO / 15 and Tgfbl. In terms of the WNT pathway, LCs were enriched for WNT antagonists Dkk3, S rp 1 / 2 '5, Wifi, while rib chondrocytes for WNT ligands Wnt5a / 5b / 10b / l 1. Rib chondrocytes also prominently expressed Ihh, Sppl, Tn Vegfa and a large number of chemokines Ccl2 / 3 / 4 / 5 / 7 / 8 / 9 / 12 / 24, Cxcll / 2 / 3 / 10 / 16 and interleukins Illb / lf9 / 17b / 17d. Other secreted signals specifically enriched in LCs include angiopoietins Angpt4, Angptl7, FGF ligands Fgf7 and Fgfl8, IGF binding proteins Igfbp2 / 4 / 5 / 6 / 7 and cytokine-like protein Cytll.

[0071] LCs and rib chondrocytes showed distinct expression patterns for transcriptional regulators, including homeobox and homeobox-like genes. Reflecting their thoracic origin, rib chondrocytes were enriched for Hoxa3.'576 / 7, Hoxb2 / 7, and Hoxc5 / 6 / 8. In a similar fashion, ear LCs were enriched for Hoxa2, Dlx5 / 6, and Msxl, reflecting their origin from neural crest mesenchyme of the second pharyngeal arch. Both cartilage cell types shared expression of known chondrogenic regulators, including Id2, Smad4, Snail '2, Sox5 / 6 / 8 / 9, and Twistl. At the same time, only rib chondrocytes expressed high levels of the followingestablished regulators of chondrogenesis: Foxa3, Foxo4, Nkx3-2, RunxI / 3, Smcid7, Sox4, Sp7, and Vdr. LCs specifically expressed Cbx6, Foxn3, Glis2 / 3, Lhx8, Mkx, Sex, Tbxl5. Notably, they also expressed several known transcriptional regulators of adipogenesis: Cebpa, Klf9, Nfia, Pparg, Srebfl and Zfp423, albeit at lower levels than adipocytes.

[0072] Spatial transcriptomic data analysis. The inventors examined recently published high-resolution spatial sequencing data (Stereo-seq technology) collected on sagittal sections cut through the whole mouse embryo at developmental stage E16.5. Specifically, the inventors analyzed the ‘E16.5_E2S5.MOSTA’ sample, which was downloaded from https: / / db.cngb.org / stomics / mosta / download / , as it was significantly enriched for cartilage markers in the head regions. In the original study, spot counts were normalized to the median of total counts across all spots and then log-transformed, adding a pseudocount of 1. The inventors analyzed the head and tail regions separately. First, the inventors extracted the x coordinates and y coordinates of the spatial spots from the entire tissue, which the inventors denote as X and Y, respectively. The inventors reflected Y i-> — Y to orient the tissue section in the correct direction when plotting X and Y directly. To isolate cartilage spots in the head or tail, the inventors filtered for spots that were annotated in the original study as either ‘Cartilage’ or ‘Cartilage primordium’ and whose coordinates satisfied X < —200 and — 314 < Y < —255 (head cartilage) or X < —340 and Y < —500 (tail cartilage). The inventors then translated the spots from the tail region in the positive vertical direction so that they were closer to the spots from the head region. The inventors reannotated the subsetted tissue as follows. Spots from the tail region that were originally annotated as ‘Cartilage’ were reannotated as ‘Tail’. Spots from the head region that were originally annotated as ‘Cartilage primordium’ were reannotated as ‘Nasal cavity’. Spots from the head region that were originally annotated as ‘Cartilage’ and whose locations satisfied Y > —350 (to avoid the tail cartilage) and Y < — 1.915(A + 370) were reannotated as ‘Nasal’ . All other spots from the head region that were originally annotated as ‘Cartilage’ were reannotated as ‘Palate’.

[0073] For quantitative real-time PCR (qRT-PCR), cDNA from hESC-derived cartilage pellets was generated from RNA extracted as indicated above using Reverse Transcriptase II cDNA synthesis kit (Invitrogen) and Oligo(dt)20 primer (ThermoFisher). qRT-PCR was performed on a QuantStudio7 instument (ThermoFisher) using manufacturer’s recommendations .

[0074] Modified diet experiments. Male C57BL / 6J mice were fed ad libitum with a high fat chow (for 39 weeks, starting at P21, or placed under caloric restriction for 72 hours with water only starting at P33. Control mice were fed ad libitum with normal chow for 39 weeks starting at P21. The obesity model mice C57BL / 6J DIO were purchased from JAX at 16 weeks of age, following 12 weeks of high fat diet.

[0075] Fluorescent free fatty acid incorporation. 100 pl of 20 pmolar Cl-BODIPY- C12 were injected peritoneally. Adult one-month old mice received single injection and were chased for 6 hours. P10 mice received daily injections at 24-hour interval for eight days, and were chased for 6 hours after the last injection.

[0076] Histological measurements. Cell area and ear pinna area was measured in ImageJ using the polygon selection tool and the Measure function. Ear pinna area measurements were standardized using natural ear landmarks (apex of the antihelix) for all samples. Lipid droplet area was measured using the MRI Lipid Droplet Tool.

[0077] Topical drug treatment. C75 (Cayman Chemical), ND-646 (MedChemExpress), and PF 05175157 (TOCRIS) were dissolved individually in DMSO at 40 mmolar, 5 mmolar, and 50 mmolar concentrations, respectively. 20 pl of either drug were painted on the ears of WT mice for 10 consecutive days, starting at Pl 1. Changes to ear morphology were monitored daily, and final size was measured on day 10 of treatment, at P21. Control mice received topical treatment of DMSO vehicle only (VWR).

[0078] Glucose modulation experiments. Culture media was prepared with glucose-free DMEM, 10% FBS (ThermoFisher), and IX PenStrep. Media was supplemented with either low (4 mM) or high (25 mM) glucose (Gibco). Day Pl 6 mouse ears were micro dissected by removing both caudal and rostral skin flap and trimming away excess muscle tissue from the cartilage and rinsing in fresh IX PBS. Tissues were then cultured for 5 days in low or high glucose conditions. Media was replaced every 48 hours. Tissues were cultured at 37 °C in 5% CO2, 5% 02 throughout the procedure.

[0079] Glucose-d7 ex vivo ear culture. P14 ears were partially dissected by removing the caudal skin flap and exposing the cartilage, then cultured in media supplemented with glucose-d7. Briefly, glucose-d7 containing media was prepared with glucose-free DMEM (ThermoFisher Scientific), 25 mM glucose-d7 (Cambridge Isotope Laboratories), 10% FBS (Atlanta Biologicals), and IX PenStrep. Tissues were cultured for 72 hours. Control sampleswere cultured for 72 hours in high glucose (25 mM) DMEM, 10% FBS, IX PenStrep without glucose-d7 added. Media was replaced every 24 hours. All samples were rinsed with IX PBS twice for 5 minutes and fixed with 4% PFA for 1 hour and imaged. Tissues were cultured at 37 °C in 5% CO2, 5% 02 throughout the procedure.

[0080] hESC-derived cartilage pellet generation. hESCs were differentiated into neural crest intermediates and then terminally into cartilage pellets. Briefly, Human embryonic stem cells line Hl (WA01) (WiCell Research Institute, Inc) was maintained in mTeSR 1 (Stem Cell Technologies) on matrigel coated dishes at 37 °C in 5% CO2, 5% 02 and passaged regularly. For hNC differentiation, cells were collected 5 days after the last passage at ~80%- 90% confluency. Cells were first rinsed 3 times in IX Ca+ and Mg + free PBS, then dissociated in Accutase (Stem Cell Technologies) for 4 min 30 s at 37 °C in 5% CO2, 5% 02. Accutase was immediately quenched with warmed IX DMEM / F12 (Invitrogen) containing 10 pM Rock Inhibitor, Y-27632 (Tocris), then centrifuged 1200 RPM for 4 min at room temperature. Media was discarded without disturbing cell pellets and replaced with NC Induction media [IX DMEM / F12 (Invitrogen), IX serum-free B27 supplement (Invitrogen), IX Glutamax (ThermoFisherScientific), 0.5% BSA (Sigma)] containing 10 pM Y-27632. hESC pellets were further dissociated to single cells mechanically through a 5 mL serological pipette. CHIR99021 (Tocris) was added to pellets in induction media during dissociation to a final concentration of 3 pM. Cells were counted and diluted to an optimal seeding density of 20xl03cells / cm2per vessel, then seeded on hESC-qualified Matrigel (BD) pre-coated vessels. 10 pM Rock Inhibitor, Y-27632, was added from days 0-2, then left out of induction media during the remainder of culture. Induction media was changed daily until day 5, when terminal differentiation began. For terminal differentiation into cartilage pellets, hESC-derived NC intermediates were centrifuged as described above to generate cell pellets. Pellets were carefully rinsed and left intact, and media was replaced with Mesencult-Chondrogenic Differentiation media (Stem cell technologies). At terminal differentiation day 21, pellets were gently dislodged from the bottom of the tube but not dissociated. Media was replaced every 2- 3 days until collection for analysis. Cells were cultured at 37 °C in 5% CO2, 5% 02 throughout the procedure.

[0081] SRS microscopy. Ex vivo cultured ears were imaged through stimulated Raman scattering to visualize lipid content and track deuterium incorporation into lipiddroplets. Stimulated Raman loss was measured using a combination of a fixed stokes beam at 1064 nm generated by a 76-MHZ mode-locked Nd: Vanadate laser (Picotrain, High-Q) delivering 7 picosecond pulses and a variable pump beam generated through an optical parametric oscillator (OPO, Levante Emerald, APE) pumped by the second harmonic of the laser at 532 nm. For imaging of the CH2 lipid mode, the OPO was tuned to 817 nm and for deuterated imaging it was tuned to 868 nm. For stimulated Raman loss, the stokes beam was modulated at 10 MHz using an acousto-optic modulator (AOM, Crystal Technology). After modulation, the two beams were combined and directed into a laser scanning inverted microscope (Fluoview 300 & 1X71, Olympus). The SRS signal was collected on an output channel of the lock-in by the synced Fluoview system to form the intensity image. Additionally, linear Raman spectra were acquired for the P 14 ear samples cultured for 72 hours and for pure standards using a confocal Raman microscope (In Via Confocal, Renishaw).

[0082] Fluorescence lifetime imaging microscopy (FLIM). Adult one-month-old mouse ear cartilage plates were microdissected, rinsed in IX PBS, and mounted in microscope slides. Inguinal adipose tissue was rinsed in IX PBS, cut into 5 mm squares, and mounted. All dissections were performed within 1 hour of imaging. All FLIM data acquisition and processing were performed with software developed at the Laboratory of Fluorescence Dynamics (University of California, Irvine).

[0083] Electron microscopy. Human fetal tissue samples (ear and nasal septum cartilage, GW20-21) were fixed in 10% phosphate buffered (pH 7.2-7.4) formalin for 2-3 days at room temperature, and 1.5 mm2 pieces were cut out from each sample. Samples were subjected to three 10 minute washes in 0.1M Millonig’s phosphate buffer and fixed by immersion in 1% osmium tetroxide fixative (SIGMA-ALDRICH) in 0.1M Millonig’s phosphate buffer (pH 7.4) containing 5.4 % glucose at 4 0C for 1 hour, dehydrated in graded dilutions of ethanol, followed by an ethanol -acetone mixture and, lastly, through three steps of acetone before embedding in Araldite. Sections were cut with a Leica EM UC7 ultramicrotome using glass knives and mounted on formvar-film coated copper grids. Thin sections from preselected areas were stained by immersion in a saturated solution of uranyl acetate and lead citrate Sections were examined with a JEM-1011 electron microscope at various magnifications.

[0084] Super-resolution microscopy. Structured illumination microscopy (aka super-resolution) images were taken using an Elyra 7 (Zeiss) with a Plan-Apochromat 63x / 1.4 Oil DIC M27 objective.Example 11: Sources of lipocartilage progenitor cells and in vitro expansion

[0085] Lipocartilage progenitor cells, aka LC progenitors, may be sourced from pre-existing craniofacial or skeleton cartilage tissues such as ear, nose, rib and xiphoid, and articular cartilage to expand their numbers in vitro. Following biopsy, cartilage tissues are dissociated in DMEM containing Trypsin-Collagenase D (0.025% to 2%) 37°C in 5% CO2, 5% 02 until single cells are evident, which may benefit from overnight digestion. Enzyme activity is quenched by adding a 5% volume of FBS. Cells are centrifuged at 300G for 4 minutes at room temperature. And rinsed twice with 1ml IX PBS (Ca+ and Mg+ free) with 10% FBS. Cells are then counted on a hemocytometer and diluted to an optimal seeding density of 20 X 103cells / cm2per vessel, and then seeded on culture vessels for expansion in chondrogenic culture medium (aka CCM), containing DMEM with high glucose / GlutaMAX, Penicillin-Streptomycin-Fungizone, non-essential aminoacids, L-proline, sodium pyruvate, and dexamethasone, and supplemented with fetal bovine serum, TGF-pi, bFGF, and PDGF. Cells will be maintained at 37°C in 5% CO2, 5% 02 for a minimum of 5 days, with frequent media replacement (2-3 days). LC progenitor cells can be passaged or immediately used for downstream lipocartilage differentiation.Example 12: Neural crest-identity induction for human pluripotent stem cells

[0086] Human pluripotent cells, HPCs (either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs)) may be maintained in mTeSRl (Stem Cell Technologies) on matrigel coated dishes at 37°C in 5% CO2, 5% 02 and passaged regularly with Dispase or Versene. To differentiate HPCs into a neural crest (NC) intermediate, HPCs are collected 4 days after the last passage at 80% to 90% confluency. Cells are rinsed 3 times in IX PBS (Ca+ and Mg+ free), then dissociated in Accutase for 4 minutes 30 seconds at 37°C in 5% CO2, 5% 02. Accutase activity is quenched by adding 1XDMEM / F12 containing lOuM Rock Inhibitor Y-27632, then cells are centrifuged at 300 g for 4 minutes at room temperature. Without disturbing the pellet, supernatant is discarded and replaced with NC Induction media [1XDMEM / F12, IX serum-free B27 supplement, IX Glutamax, 0.5% BSA] containing lOuMY-27632. CHIR99021 is added to a final concentration of 3uM and HPC clusters are dissociated to single cells mechanically by aspiration through a 5mL serological pipette a total of 20 times. Cells are then counted on a hemocytometer and diluted to an optimal seeding density of 20 X 103cells / cm2per vessel, and then seeded on culture vessels precoated with Matrigel. From days 0-2 of culture, lOuM Rock Inhibitor Y-27632 is added. Cells are maintained in induction media for 2-5 days (induction efficiencies may change based on cell sources, media lots and providers, etc.). Induction media is changed daily until day of collection for further analysis. Cells are cultured at 37°C in 5% CO2, 5% 02 throughout the procedure.Example 13: Other autologous cell sources for lipocartilage cell identity-induction

[0087] In addition, other autologous cell sources of lipocartilage progenitors may include adipose tissue-derived stem cells from the stromal vascular fraction following liposuction, bone marrow-derived mesenchymal stem cells, and peripheral blood-derived mesenchymal stem cells. Such progenitors will benefit from large-scale expansion before use, in either a) bioreactors or; b) spinner flasks or; c) roller bottles or; d) multilayered flasks; in specialized media such as otMEM supplemented with FBS and bFGF, and maintained at 37°C in 5% CO2, 5% 02 for a minimum of 3 days.Example 14: Lipocartilage differentiation of progenitor cells

[0088] Following expansion, lipocartilage progenitors are centrifuged in 15 ml polystyrene tubes at 150 X g for 5 minutes in CCM. Following pellet formation, media is replaced with Differentiation media consisting of CCM, which may also contain a combination of factors to sustain chondrogenic maturation, including but not limited to: BMP1, BMP2, BMP5, GDF5, GDF10, IGF1, IGF2, IGFBP2, IGFBP4, IGFBP5, IGFBP6, IGFBP7, TGFB3, DKK3, SFRP1, SFRP2, SFRP5, WIFI, ANGPT4, ANGPTL7, FGF7, FGF18, and CYTL1, and other factors listed in our Specifications. Pellets are maintained at 37°C in 5% CO2, 5% 02 for a minimum of 21 days. Tube caps are left on loose to allow for air flow. -90% of the media is replaced every 2 days.Example 15: Method for in vitro maturation of lipocartilage cells

[0089] Lipocartilage pellet size increases with time, and after 7-10 days pellets become visible with the naked eye. On day 10, pellets are gently dislodged by carefully pipetting media around each pellet until they float free. Lipocartilage pellets can be evaluated for lipid vacuoles after day 21.Example 16: Single cell suspension preparation for lipocartilage cells

[0090] Lipocartilage pellets should be transferred onto a 50 mm Petri dish with 3- 4 ml of IX PBS (Ca+ and Mg+ free) containing 0.5 mg / mL Collagenase II and triturate using a scalpel blade. Pellets should be minced into 1 mm pieces or smaller if possible. Resulting slurry should be transferred into a 15 ml polystyrene tube and incubated at 37°C in a water bath with frequent pipetting for a minimum of 30 minutes. Dissociation efficiency should be evaluated using a hemocytometer (>90% single cells). Collagenase activity should be quenched by adding up to 10% FBS and filter twice through a 100 um cell strainer.Example 17: Lipid vacuole labeling of mature lipocartilage cells

[0091] Single cells should be pelleted by centrifugation at 300 x g at 4°C for 10 minutes. Supernatant should be removed, and pellet should be dissociated in 1ml IX PBS (Ca+ and Mg+ free) with 10% FBS and supplemented with a non-toxic fluorescent lipid dye (for example, 0.001% BODIPY 493 / 503) or 1ml IX PBS (Ca+ and Mg+ free) with 10% FBS and the optimal concentration of alternative fluorescent lipid dye, according to manufacturer’s instructions. Cells should be incubated on ice and protected from light for a minimum of 30 minutes. Single cells should then be pelleted by centrifugation at 300 x g at 4°C for 10 minutes. Cells should be rinsed pellet dissociated in 200 ul IX PBS (Ca+ and Mg+ free) with 10% FBS.

[0092] Other additional examples of non-toxic fluorescent lipid dyes for possible use include but are not limited to BODIPY 500 / 510, BODIPY 530 / 530, BODIPY 558 / 568, BODIPY 576 / 589, BODIPY 581 / 591, LipiDye, LipidGreen, LipidGreen2, LipidTox, SMCy3, SMCy3.5, SMCy5, SMCy5.5, SMCy7, or SMCy7.5.Example 18: Purification of lipid vacuole-containing lipocartilage cells

[0093] Single, lipid vacuole-containing lipocartilage cells can be purified by physical methods such as buoyancy or biochemistry-based methods such as fluorescenceactivated cell sorting (FACS). In one example, buoyancy separation consists of centrifugation at 150 x g for 5 minutes at 4C. Cells within the buoyant fraction can be collected by pipetting. Alternatively, single cells can be stained with lipid dyes such as BODIPY 493 / 503 as well as fluorescent viability dye such as propidium iodide (PI) prior to purification, to detect LV- containing as well as dead cells, respectively, in the population to be purified. Single cells should be selected using Forward versus Side scatter gating. Pl-stained cells are considered dead, and only the Pl-negative fraction should be used. BODIPY 493 / 503-positive subset of Pl-negative cells should then be isolated and retained. These represent mature lipid vacuolecontaining human lipocartilage cells with head and neck identity.Example 19: In vitro maturation of purified lipocartilage cells

[0094] FACS-purified lipid-containing lipocartilage cells (aka LCs) can then be further matured for an extended time by transferring them up to 20 X 103cells / cm2vessel and centrifuging at 300 x g for 10 min at room temperature to form a new pellet. The supernatant can be discarded and replaced with pre-warmed IX DMEM / F12 (25 mM glucose) with 10% FBS without disturbing the pellet. Cell pellets can be maintained for up to 7 days. The rate of lipid droplet growth can be modulated by increasing or decreasing glucose concentration in the media accordingly, as well as addition of known lipogenic factors such as insulin and IGF1.Example 20: Synthetic extracellular matrix (ECM) generation

[0095] Synthetic ECM scaffolds based on naturally-occurring proteins in head and neck (aka craniofacial) cartilage can be prepared in the laboratory. Craniofacial cartilage cell pellets express a variety of structural ECM proteins in a signature ratio. To generate an “craniofacial ECM” the inventors will fabricate a matrix-like biomaterial composed of the most abundant ECM proteins found in human craniofacial cartilage pellets. Top ECM proteins are: Collagen VI, Collagen VII, Collagen VIII, Collagen IX, Collagen X, Collagen XI, Myocilin, Fibronectin, Aggrecan, Tenascin, Thrombospondin. “Craniofacial ECM” hydrogel constructs can be prepared from different ratios of the above listed purified proteins and in different combinations or by themselves. Several cross-linking strategies are available to fabricate 3D structures from ECM proteins, including 1,4-butanediol diglycidyl ether (BDDGE) or genipin cross-linking. Further, crosslinked collagen-chitosan gels can be also used to generate synthetic ECM scaffolds to resemble human craniofacial cartilagebiomechanics. Synthetic ECM constructs can be made in custom shapes to generate a “preformed” tissue to mimic the patient’s natural shapes and facilitate grafting.Example 21: Generation of living-tissue lipocartilage constructs

[0096] Living-tissue lipocartilage constructs can be generated by combining FACS-purified lipid-containing lipocartilage cells with synthetic ECM constructs. Purified single cells can be seeded onto a previously prepared synthetic ECM scaffold and maintained in vitro.Example 22: Grafting of craniofacial lipocartilage pellets, single cells, or living-tissue constructs

[0097] Craniofacial reconstructive surgery commonly involves grafting of autologous or allogenic cartilage tissues from non-craniofacial sites. The latter is its major limitation. The inventors propose using HPCs-derived purified lipid-containing craniofacial lipocartilage cells as a source to fabricate lipocartilage and lipocartilage-like materials for: (a) grafting of cell pellets, (b) grafting of individual cells in suspension, (c) grafting of living lipocartilage-tissue constructs, or (d) a combination approach of a, b and c above.Example 23: FACS purification protocol using BODIPY 493 / 503 as a lipophilic dye.

[0098] For mouse tissues, animals at postnatal day 13 are euthanized and skin tissues collected. Microdissected ear cartilage is placed in freshly-prepared saline (PBS) and cut into lmm2 pieces. Cartilage pieces are collected and incubated in 0.1% Collagenase D in PBS for 1-3 hours at 37°C with gentle rocking. One resulting three-dimensional rendering of BODIPY-stained microdissected ear cartilage from one-month-old wild type mouse is as shown in FIGs. 3A-3D.

[0099] For human cartilage organoids, tissues are minced into small pieces roughly lmm3. hESC-derived cartilage organoids contain abundant lipids. Following 44 days in maturation media, hESC-derived cartilage organoids are stained with BODIPY to reveal abundant lipid vacuoles. hESC-derived can be purified using lipid vacuole marker. Following tissue dissociation, Day 44 cartilage pellets are stained with BODIPY to stain lipid fluorescently. Such cells are ready for purification using fluorescence-activated cell sorting(FACS). Upon reaching the desired digestion level, the enzymatic reaction is stopped by adding a 5% volume of BSA. Undigested large pieces and debris can be removed by passing cells through a 40pM strainer. Cells are centrifuged at 500 g for 5 minutes and the pellet rinsed with fresh IX PBS. Cells are resuspended in 1 ml of IX PBS with 0.1% BODIPY 493 / 503 and incubated for 15 minutes at 37°C. Cells are centrifuged at 500 g for 5 minutes and the pellet rinsed with fresh IX PBS. Cells are resuspended in 1 ml of IX PBS with 1% bovine serum albumin (FBS). Lipid-filled cells can be sorted by FACS by selecting single cells (linear FSC- A vs FSC-H) with distinct intensity values along the FITC channel (BODIPY stain). Further example purification and in vitro growth strategies for hESC-derived cartilage organoids are as shown in FIG. 4.Example 24: In vitro growth of lipid-containing cartilage organoids.

[0100] Following tissue dissociation, postnatal day 13 (P13) LCs are stained with DAPI and BODIPY to stain nucleic acids and lipid, respectively. Such cells are ready for purification using fluorescence-activated cell sorting (FACS). FACS-purified cells can be pelleted into 15 ml conical tubes using as few as IxlO3cells by centrifugation at 500g for 5 minutes. Cells are rinsed with fresh IX PBS without dissociating the pellet. Up to 2 ml of freshly prepared media containing DMEM with 25 mmolar glucose, 10% FBS, and IX antimycotic / antibiotic should be carefully added to each pellet and incubated at 37°C, 5% CO2. Up to 90% of media should be replaced every other day with pre-warmed media of the same type taking care not to disturb the pellet. Lipid-fdled mouse cartilage organoids are evident by 7 days by positive BODIPY staining. Further example purification and in vitro growth strategies for mouse LCs are as shown in FIGs. 5A-5B.Terms

[0101] In the present disclosure, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in this disclosure, including the drawings and claims, are not meant to be limiting. Some embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the presentdisclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0102] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs when read in light of the current disclosure.

[0103] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0104] The articles “a” and “an” are used herein to refer to one or to more than one (for example, at least one) of the grammatical object of the article, unless the context dictates otherwise. By way of example, “an element” means one element or more than one element.

[0105] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is approximately the recited value. Where it is not clear from the context what is encompassed by “about,” it will mean the value recited + / - 10%.

[0106] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0107] The terms “individual”, “subject”, or “patient” as used herein have their plain and ordinary meaning as understood in light of the specification, and mean a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a nonhuman primate, or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate. Theterm “mammal” is used in its usual biological sense. Thus, it includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like.

[0108] As used herein, the term “isolated” has its plain and ordinary meaning as understood in light of the specification, and refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from equal to, about, at least, at least about, not more than, or not more than about, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated (or ranges including and / or spanning the aforementioned values). In some embodiments, isolated agents are, are about, are at least, are at least about, are not more than, or are not more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and / or spanning the aforementioned values). As used herein, a substance that is “isolated” may be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” may refer to a cell not contained in a multi-cellular organism or tissue.

[0109] As used herein, “in vivo” has its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method inside living organisms, usually animals, mammals, including humans, and plants, as opposed to a tissue extract or dead organism.

[0110] As used herein, “ex vivo” has its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside a living organism with little alteration of natural conditions.[oni] As used herein, “in vitro” has its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside of biological conditions, e.g., in a petri dish or test tube.

[0112] The term “purity” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refersto the actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity may be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membrane, cell debris, small molecules, degradation products, solvent, carrier, vehicle, or contaminants, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents. Purity can be measured using technologies including but not limited to electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectrometry, infrared spectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0113] The term “yield” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the actual overall amount of the substance, compound, or material relative to the expected overall amount. For example, the yield of the substance, compound, or material is, is about, is at least, is at least about, is not more than, or is not more than about, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected overall amount, including all decimals in between. Yield may be affected by the efficiency of a reaction or process, unwanted side reactions, degradation, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material during any step of the production.

[0114] The terms “effective amount” or “effective dose” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to that amount of a recited composition or compound that results in an observable effect. Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that is effective to achieve the desired response for a particular subject and / or application. The selected dosage level will depend upon a variety of factors including, but not limited to, the activity of thecomposition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0115] The terms “function” and “functional” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to a biological, enzymatic, or therapeutic function.

[0116] The term “inhibit” as used herein has its plain and ordinary meaning as understood in light of the specification, and may refer to the reduction or prevention of a biological activity. The reduction can be by a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values. As used herein, the term “delay” has its plain and ordinary meaning as understood in light of the specification, and refers to a slowing, postponement, or deferment of a biological event, to a time which is later than would otherwise be expected. The delay can be a delay of a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the aforementioned values. The terms inhibit and delay may not necessarily indicate a 100% inhibition or delay. A partial inhibition or delay may be realized.

[0117] As used herein, the terms “treating” or “treatment” have their plain and ordinary meaning as understood in light of the specification, and refer to an approach for obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (e.g., not worsening) the state of disease, prevention of a disease's transmission or spread, delaying or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the recurrence of disease, and remission, whether partial or total and whether detectable or undetectable. “Treating” and “treatment” as used herein also includeprophylactic treatment. Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administering step may include a single administration or may include a series of administrations. The compositions are administered to the subject in an amount and for a duration sufficient to treat the subject. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age and genetic profile of the subject, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required.

[0118] The term “administering” includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intra-tumoral, intrathecal, intranasal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intra-tumoral, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “coadminister” it is meant that a first compound described herein is administered at the same time, just prior to, or just after the administration of a second compound described herein.

[0119] As used herein, “pharmaceutically acceptable” has its plain and ordinary meaning as understood in light of the specification and refers to carriers, excipients, and / or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity. A “pharmaceutically acceptable” “diluent,” “excipient,” and / or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and / orcarrier is a diluent, excipient, and / or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs. The term diluent, excipient, and / or carrier can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical formulation is administered. Such pharmaceutical diluent, excipient, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include sugars, starch, glucose, fructose, lactose, sucrose, maltose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, salts, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution. The physiologically acceptable carrier may also include one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, isomalt, maltitol, or lactitol, salt-forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The formulation, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These formulations can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode of administration.

[0120] The term “pharmaceutically acceptable salts” has its plain and ordinary meaning as understood in light of the specification and includes relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such asethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di- , and triethanolamine; amino acids, including glycine, arginine and lysine; guanidine; N- methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; trihydroxymethyl aminoethane.

[0121] The term “% w / w” or “% wt / wt” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100. The term “% v / v” or “% vol / vol” as used herein has its plain and ordinary meaning as understood in the light of the specification and refers to a percentage expressed in terms of the liquid volume of the compound, substance, ingredient, or agent over the total liquid volume of the composition multiplied by 100.

[0122] The terms “first,” “second,” and “third” used in combination with substances are intended to designate distinguishable features to similar substances and do not imply any particular order unless otherwise specified.

[0123] The terms “LV”, “lipid vacuole,” “lipid droplet,” or “lipid vesicle” all have the same meaning, and refer to a single-layered membrane vesicle within the cytoplasm of a cell containing neutral lipid species within. As disclosed herein, these lipid vacuoles are found within the cytoplasm of adult lipocartilage cells, LCs.

[0124] Although the present disclosure has been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of embodiments of the disclosure as defined in the appended claims.

[0125] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can beeasily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 markers refers to groups having 1, 2, or 3 markers . Similarly, a group having 1-5 markers refers to groups having 1, 2, 3, 4, or 5 markers, and so forth.

[0126] All patent filings, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. Any feature, step, element, embodiment, or aspect disclosed herein can be used in combination with any other unless specifically indicated otherwise.

[0127] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) 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.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases ”at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a“ or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as ”a“ or ”an“ (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize thatsuch recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a formulation having at least one of A, B, and C” would include but not be limited to formulations that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a formulation having at least one of A, B, or C” would include but not be limited to formulations that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc ). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0128] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0129] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. For example, “about 5”, shall include the number 5. Finally, as will be understood by one skilled in the art, a range includes eachindividual member. Thus, for example, a group having 1-3 values refers to groups having 1, 2, or 3 values. Similarly, a group having 1-5 values refers to groups having 1, 2, 3, 4, or 5 values, and so forth.

[0130] It would be understood that the various sizes, materials, configurations and arrangements disclosed herein may be combined and constructed in any way that is feasible to create a hybrid for any particular end use. Accordingly, all suitable modifications and equivalents may be resorted to falling within the scope of the appended claims. Unless defined otherwise, all technical and scientific terms used herein have same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Also, as used herein and in the appended claims, the singular form “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise.

[0131] It is to be understood that the present invention is not to be limited to the exact description and embodiments as illustrated and described herein. To those of ordinary skill in the art, one or more variations and modifications will be understood to be contemplated from the present disclosure. Accordingly, all expedient modifications readily attainable by one of ordinary skill in the art from the disclosure set forth herein, or by routine experimentation therefrom, are deemed to be within the true spirit and scope of the invention as defined by the appended claims.

[0132] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHA T IS CLAIMED IS:

1. A method of generating an adult lipocartilage cell, the method comprising incubating a progenitor cell capable of differentiating into the adult lipocartilage cell with cartilage-inducing media for at least 20 days.

2. The method of claim 1, wherein the progenitor cell is a stem cell, optionally wherein the progenitor cell is a pluripotent and / or induced pluripotent stem cell.

3. The method of claim 1 , wherein the progenitor cell is continuously shaken, agitated, swirled, or any combination thereof, during incubation.

4. The method of claim 1, wherein the cartilage-inducing media is chondrogenic culture and / or differentiation media, optionally wherein the cartilage-inducing media is MesenCult™-ACF Chondrogenic Differentiation Medium for MSCs.

5. The method of claim 4, wherein the chondrogenic culture and / or differentiation media comprises at least one of: DMEM, high glucose / GlutaMAX, Penicillin, Streptomycin, Fungizone, an amino acid, L-proline, pyruvate, dexamethasone, fetal bovine serum, TGF-bl, bFGF, PDGF, or any combination thereof.

6. The method of claim 5, wherein the chondrogenic culture and / or differentiation media comprises at least one of: BMP1, BMP2, BMP5, GDF5, GDF10, IGF1, IGF2, IGFBP2, IGFBP4, IGFBP5, IGFBP6, IGFBP7, TGFB3, DKK3, SFRP1, SFRP2, SFRP5, WIFI, ANGPT4, ANGPTL7, FGF7, FGF18, CYTL1, or any combination thereof.

7. The method of claim 1, wherein the progenitor cell originates from autologous ear cartilage, autologous nasal cartilage, autologous rib cartilage, autologous xiphoid cartilage, autologous articular cartilage, non-cartilage mesenchymal cells, adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, peripheral blood- derived mesenchymal stem cells, or any combination thereof.

8. A method of identifying an adult lipocartilage cell from a population of cells, the method comprising: screening for the presence of a lipid droplet (also referred to as a lipidvacuole, or “LV”) within a cell, wherein the presence of a lipid droplet indicates that the cell is the adult lipocartilage cell.

9. The method of claim 8, wherein the screening is performed through fluorescent activated cell sorting (FACs) and / or through a buoyancy assay, wherein the presence of the lipid droplet in the adult lipocartilage cell results in a different cellular buoyancy compared with other cells in the population of cells.

10. The method of claim 9, wherein the buoyancy assay is centrifugation.

11. The method of claim 8, wherein the presence of the lipid droplet is determined by positive staining from a dye.

12. The method of claim 11, wherein the dye is a fluorescent lipid dye and / or a BODIPY dye.

13. The method of claim 12, wherein the dye is selected from: BODIPY 493 / 503, BODIPY 500 / 510, BODIPY 530 / 530, BODIPY 558 / 568, BODIPY 576 / 589, BODIPY 581 / 591, LipiDye, LipidGreen, LipidGreen2, LipidTox, SMCy3, SMCy3.5, SMCy5, SMCy5.5, SMCy7, SMCy7.5, or any combination thereof.

14. A method of generating a population of adult lipocartilage cells, the method comprising: screening a general population of cells for the presence of a lipid droplet within a cell; purifying the cell comprising the lipid droplet, thus isolating an adult lipocartilage cell; and expanding the adult lipocartilage cell, thus forming the population of adult lipocartilage cells.

15. The method of claim 14, wherein the expansion is performed by incubating the adult lipocartilage cell in the same media that the general population of cells was first grown in.

16. The method of claim 14, wherein the expansion is performed for up to two weeks.

17. The method of claim 14, wherein the expansion comprises administering a media with at least one of: insulin, IGF1, BMP1, BMP2, glucose, or any combination thereof.

18. The method of claim 14, wherein the glucose is present at about 5 mM, 7 mM, 10 mM, 15 mM, 25 mM, or any integer that is between about 5 and about 25 mM.

19. The method of claim 14, wherein the expansion is performed in a bioreactor, spinner flask, roller bottle, multilayered flask, or any combination thereof.

20. A composition for use as a synthetic extracellular matrix (ECM) gel, the composition comprising at least one of: Collagen VI, Collagen VII, Collagen VIII, Collagen IX, Collagen X, Collagen XI, Myocilin, Fibronectin, Aggrecan, Tenascin, Thrombospondin, or any combination thereof.

21. The composition of claim 20, further comprising chitosan.

22. A method of generating a replacement tissue construct, the method comprising seeding the population of adult lipocartilage cells generated from the method of claim 14 with the composition of claim 20.

23. A replacement tissue construct with use in in grafting onto a subject, comprising the lipid droplet-containing adult lipocartilage cell generated using the method of claim 1.

24. The replacement tissue construct of claim 23 for use as part of replacement and / or reconstructive surgery in a subject in need thereof.

25. The replacement tissue construct of claim 23, wherein the subject has a head and / or a neck cartilage defect.

26. The replacement tissue construct of claim 25, wherein the head and / or neck cartilage defect is a defect in nose, ear, laryngeal, or tracheal cartilage, or any combination thereof.

27. The replacement tissue construct of claim 23, wherein the replacement tissue construct provides structural support of non-cartilage craniofacial tissues, skin, muscle, or any combination thereof, in the subject.

28. A method of grafting the adult lipocartilage cell generated using the method of claim 1 into a subject in need thereof, the method comprising administering the adult lipocartilage cell as part of a suspension or a cell pellet.

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