Efficacy marker identification for cartilage donor cell selection

Abstract

Described herein are fabricated cell sheet compositions comprising: a population of juvenile cartilage-derived chondrocytes (JCCs) with low gene expression of asporin (ASPN). Also described are methods for maintaining low gene expression of asporin (ASPN) in subcultured JCCs for formation of cell sheets.

Description

[0001] EFFICACY MARKER IDENTIFICATION FOR CARTILAGE DONOR CELL SELECTION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 562,141 , filed on March 6, 2024, which is incorporated by reference herein in its entirety.

[0004] BACKGROUND

[0005] Chondral defects in human knees are prevalent in both general and athletic populations from multiple etiologies. Frequently incurred with osteological disorders from acute trauma, increasing participation in athletic activities is especially associated with increasing prevalence. Articular cartilage defects have a limited capacity to regenerate after injury and may lead to accelerated wear, worsening pain, and potential arthritis progression. Many methods have been described to treat chondral defects in the knee: microfracture or drilling, osteochondral autograft transfer (OAT), osteochondral allograft (OCA), autologous chondrocyte implantation (ACI), matrix- assisted autologous chondrocyte implantation (MACI), and particulated juvenile allograft. However, fibrocartilage after microfracture, fragmentation and resorption of allografts after OAT and OCA, and disturbed fusion of the regenerative cartilage and the healthy surrounding cartilage after ACI and MACI have been reported, while low product scalability is another problem.

[0006] Scaffold-free cell-dense implants, termed “cell sheets,” are routinely fabricated using temperature-responsive cell culture surfaces. These sheets preserve intercellular interactions and extracellular matrix that facilitate intimate tissue site integration, overcoming engraftment issues often reported in other tissue- and cell-based approaches.

[0007] Allogenic cell source variability is known to affect cell-based therapeutic results. Differences in secreted paracrine regenerative and immuno-modulatory factors from mesenchymal stromal cell (MSC) and juvenile cartilage-derived chondrocyte (JCC) sheets derived from different human donors have been reported. JCC sheet characteristics relevant to their regenerative potency are affected by donor variations. To improve JCC sheet therapeutic efficacy and reliability, extents to which therapeutic effects vary with human cell sourcing must be determined. Key factors that influence JCC donor variation must be elucidated to optimize donor selection criteria using relevant animal cartilage regenerative models comparing donor JCC sheet repair efficacy.

[0008] What is needed are efficacy markers for identifying cartilage donor cell selection for cartilage regenerative medicine products. SUMMARY

[0009] One embodiment described herein is a fabricated cell sheet composition, the composition comprising: a population of juvenile cartilage-derived chondrocytes (JCCs) with low gene expression of asporin (ASPN). In one aspect, the low gene expression of ASPN is below about 100 units as measured by an RNAseq DESeq2 method. In another aspect, the population of JCCs further comprises low gene expression of COL1A 1, COL1A2, or a combination thereof. In another aspect, the low gene expression of the sum of COL1A1 and COL1A2 is below about 10 * 105units as measured by an RNAseq DESeq2 method. In another aspect, the JCCs are derived from a human polydactyly resection surgery. In another aspect, the JCCs are a mixture of two or more different cell compositions.

[0010] Another embodiment described herein is a method of maintaining low gene expression of asporin (ASPN) in subcultured juvenile cartilage-derived chondrocytes (JCCs) for formation of cell sheets, the method comprising: treating the subcultured JCCs one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby maintaining low gene expression of ASPN in the subcultured JCCs for formation of cell sheets. In one aspect, the subcultured JCCs are continuously treated with the culture medium comprising bFGF. In another aspect, the subcultured JCCs are treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL. In another aspect, the method further comprises treating the subcultured JCCs with an additional agent to maintain low gene expression of ASPN.

[0011] Another embodiment described herein is a method for producing a cell sheet composition comprising chondrocytes, the method comprising: (a) collecting living cartilage tissue by scalpel under sterile conditions, wherein the cartilage tissue comprises JCCs; (b) cutting the collected cartilage tissue with scalpels into pieces; (c) collecting JCCs from the cartilage tissue through enzymatic treatment of the pieces of cartilage tissue and sub-culturing; and (d) culturing a mixture of JCCs in culture solution on a temperature-responsive polymer which has been coated onto a substrate surface of a cell culture support, wherein the temperature-responsive polymer has a lower critical solution temperature in water of 0-80 °C; (e) adjusting the temperature of the culture solution to below the lower critical solution temperature, whereby the substrate surface is made hydrophilic and adhesion of the cell sheet to the surface is weakened; and (f) detaching the cell sheet composition from the cell culture support, thereby producing a cell sheet composition.

[0012] Another embodiment described herein is a fabricated cell sheet composition having improved cartilage repair and regenerative efficacy, the composition comprising: a population of juvenile cartilage-derived chondrocytes (JCCs) comprising low gene expression of asporin (ASPN). In one aspect, the low gene expression of ASPN is below about 100 units as measured by an RNAseq DESeq2 method. In another aspect, the population of JCCs further comprises low gene expression of COL 1A1, COL1A2, or a combination thereof. In another aspect, the low gene expression of the sum of COL1A1 and COL1A2 is below about 10 * 105units as measured by an RNAseq DESeq2 method. In another aspect, the composition generates an in vivo regenerative O’Driscoll (OD) score of greater than or equal to about 22 when implanted in vivo. In another aspect, the JCCs are derived from a human donor of 3 months to 16 years of age. In another aspect, the JCCs are derived from a human polydactyly resection surgery. In another aspect, the JCCs are a mixture of two or more different cell compositions. In another aspect, the JCCs are derived from an equine (horse) donor of less than about 2 years of age.

[0013] Another embodiment described herein is a method of maintaining low gene expression of asporin (ASPN in subcultured juvenile cartilage-derived chondrocytes (JCCs) for formation of cell sheets having improved cartilage repair and regenerative efficacy, the method comprising: treating the subcultured JCCs one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby maintaining low gene expression of ASPN in the subcultured JCCs for formation of cell sheets. In another aspect, the subcultured JCCs are continuously treated with the culture medium comprising bFGF. In another aspect, the subcultured JCCs are treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL. In another aspect, low gene expression of ASPN is maintained in the subcultured JCCs up to at least 6 passages. In another aspect, the method further comprises treating the subcultured JCCs with an additional agent to maintain low gene expression of ASPN. In another aspect, the additional agent comprises an ASPN siRNA, an ASPN shRNA, an ASPN inhibitor, an agent that suppresses ASPN gene expression, or combinations thereof. In another aspect, the agent that suppresses ASPN gene expression comprises a PPARGIa inducer.

[0014] Another embodiment described herein is a method of enhancing the expansion of subcultured juvenile cartilage-derived chondrocytes (JCCs) for formation of cell sheets having improved cartilage repair and regenerative efficacy, the method comprising: treating the subcultured JCCs one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby enhancing the expansion of the subcultured JCCs for formation of cell sheets. In one aspect, the subcultured JCCs are continuously treated with the culture medium comprising bFGF. In another aspect, the subcultured JCCs are treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL.

[0015] Another embodiment described herein is a method of maintaining low gene expression of asporin (ASPN) in a culture of fabricated cell sheets comprising a population of juvenile cartilage- derived chondrocytes (JCCs) comprising low gene expression of ASPN and having improved cartilage repair and regenerative efficacy, the method comprising: treating the culture of fabricated cell sheets one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby maintaining low gene expression of ASPN in the culture of fabricated cell sheets. In another aspect, the culture of fabricated cell sheets is continuously treated with the culture medium comprising bFGF. In another aspect, the culture of fabricated cell sheets is treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL.

[0016] Another embodiment described herein is a method for producing a cell sheet composition comprising chondrocytes, the method comprising: (a) culturing a mixture of JCCs in culture solution on a temperature-responsive polymer which has been coated onto a substrate surface of a cell culture support, wherein the temperature-responsive polymer has a lower critical solution temperature in water of 0-80 °C; (b) adjusting the temperature of the culture solution to below the lower critical solution temperature, whereby the substrate surface is made hydrophilic and adhesion of the cell sheet to the surface is weakened; (c) detaching the cell sheet composition from the cell culture support, thereby producing a cell sheet composition, optionally wherein the method further comprises: (d) collecting cartilage tissue by scalpel under sterile conditions, wherein the cartilage tissue comprises JCCs; (e) cutting the collected cartilage tissue with scalpels into pieces; and (f) collecting JCCs from the cartilage tissue through enzymatic treatment of the pieces of cartilage tissue.

[0017] Another embodiment described herein is a method for repairing and / or regenerating cartilage in a subject in need thereof, the method comprising: treating the subject with a fabricated cell sheet composition comprising a population of juvenile cartilage-derived chondrocytes (JCCs) comprising low gene expression of asporin (ASPN). In another aspect, the population of JCCs comprising low gene expression of ASPN is pre-selected from a plurality of donor cell groups having different levels of ASPN gene expression. In another aspect, the subject is a human subject or an equine (horse) subject.

[0018] DESCRIPTION OF THE DRAWINGS

[0019] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0020] FIG. 1 A-C show an overview of the study and human cell sheet efficacy stratification in a nude rat chondral defect xenograft model. FIG. 1A shows a scheme for marker identification. Juvenile chondrocyte (11 donors were cultured and banked at the end of P0, then employed for cell sheet production. The created cell sheets from all donors were tested with (1) in vivo xenograft model on rat chondral defect model and efficacy of each donor-derived cell sheets was stratified by modified O’Driscoll scoring and (2) RNAseq analysis. FIG. 1 B shows representative images of macroscopic observation the knee after cell sheet transplantation (top) and Safranin O staining (bottom) at 4 weeks after transplantation. Left: more effective example, Right: less effective example. FIG. 1C shows stratification of in vivo efficacy by individual cell sheets on the chondral defect model using modified O’Driscoll scoring (n = 3-6 for each donor).

[0021] FIG. 2A-C show RNAseq analysis of cell sheets before transplantation. FIG. 2A shows a heatmap of 10 individual cell sheets using differentially expressed genes. FIG. 2B shows a volcano plot for the top significantly expressed genes; x-axis: fold change, y-axis: p-value. FIG. 2C shows a list of the top differentially expressed genes. These data are also shown in Table 2.

[0022] FIG. 3A-D show the effect of basic fibroblast growth factor on juvenile chondrocyte culture. FIG. 3A shows basic fibroblast growth factor (bFGF) effect on passage culture of juvenile chondrocytes (JCCs). FIG. 3B shows bFGF effect on colony size of JCCs in primary culture. FIG. 3C shows a comparison of chondrogenic pellet size with or without bFGF media supplementation. FIG. 3D shows a Principal Component Analysis (PCA) using Passage 2, 4, and 6 cell sheets of four donors. The PCA plot shows that culture supplement of 1.0 ng / mL bFGF shifts cell sheet properties into clusters where early-passage cell sheets are located. Cell sheets were produced from four donors: two from “more effective” donors and two from “less effective” donors, and all four were used for RNAseq samples.

[0023] FIG. 4A-B show bFGF effects on putative efficacy marker genes. Comparisons of donor differences in asporin (FIG. 4A) and PPARGC1A (FIG. 4B) gene expression with bFGF supplementation in JCC culture are shown, x-axis: sample ID, y-axis: normalized counts in DEseq2 analysis.

[0024] FIG. 5A-B show augmentation of in vivo chondrogenic regenerative effects using JCC sheets prepared with bFGF-supplemented production. FIG. 5A shows the experimental design. FIG. 5B shows the macroscopic and histological images of JCC sheet-transplanted samples harvested from nude rats. Safranin O staining for sulfated glycosaminoglycan, immunostaining for type 2 collagen, type 1 collagen, and human vimentin (n - 3 for each group) are shown.

[0025] FIG. 6 shows the validation of siRNA knock-down of PPARGC1A as the targeted gene in cell sheet culture when transplanted in vivo in the rat defect model. Resulting in vivo cell sheet transplantation on nude rat chondral defect model produced the microscopy images showing the effect of PPARGC1 A knock-down as a regulator of asporin. Left: Control siRNA condition. Right: cell sheet knocked down with PPARGC1A siRNA. Efficacy of PPARGC1A knockdown was confirmed at 4 weeks after transplantation by the presence of cartilage tissue in the defects of the PPARGC1A knock-down cell sheets (n = 2-3).

[0026] FIG. 7A-C show the correlation analysis of in vivo cartilage O’ Driscoll scores and RNAseq normalized counts in DESeq2 analysis. FIG. 7A shows a list of the most correlated genes to the modified O’Driscoll scores from 11 donor cell sheet transplantation in vivo results. FIG. 7B-C show examples of correlation between normalized counts in DESeq2 and O’Driscoll scores.

[0027] FIG. 8A-E show histological samples for bFGF-supplemented JCC sheet transplantation. FIG. 8A shows macroscopic images. FIG. 8B shows Safranin O staining. FIG. 8C shows type 2 collagen. FIG. 8D shows type 1 collagen. FIG. 8E shows human vimentin. n = 3 for each group.

[0028] DETAILED DESCRIPTION

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0030] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0031] As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of’ the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, “comprising,” is an “open- ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim.

[0032] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0033] As used herein, the term “or” can be conjunctive or disjunctive.

[0034] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0035] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0036] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.”

[0037] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”

[0038] As used herein, the terms “room temperature,” “RT,” or “ambient temperature” refer to the typical temperature in an indoor laboratory setting. In one aspect, the laboratory setting is climate controlled to maintain the temperature at a substantially uniform temperature or with a specific range of temperatures. In one aspect, “room temperature” refers a temperature of about 15-30 °C, including all integers and endpoints within the specified range. In another aspect, “room temperature” refers a temperature of about 15-30 °C; about 20-30 °C; about 22-30 °C; about 25-30 °C; about 27-30 °C; about 15-22 °C; about 15-25 °C; about 15-27 °C; about 20-22 °C; about 20-25 °C; about 20-27 °C; about 22-25 °C; about 22-27 °C; about 25-27 °C; about 15 °C ± 10%; about 20 °C ± 10%; about 22 °C ± 10%; about 25 °C ± 10%; about 27 °C ± 10%; ~20 °C, ~22 °C, ~25 °C, or ~27 °C, at standard atmospheric pressure.

[0039] As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result “Control” also refers to control experiments or control cells.

[0040] As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.

[0041] As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein.

[0042] As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.

[0043] As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject’s age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.

[0044] As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), nonhuman primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human. In another embodiment, the subject is a horse.

[0045] As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0046] As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0047] As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.

[0048] As used herein, “juvenile cartilage-derived chondrocytes” or“JCCs” refer to chondrocytes obtained from primary human juvenile tissue (age between 6 and 30 months). Typically, these chondrocytes were obtained from polydactylous digits that were amputated during routine surgery.

[0049] As used herein, “with low gene expression of asporin (ASPN)” refers to juvenile cartilage- derived chondrocytes that have low expression levels of an asporin gene (e.g., below 100 units) as measured using an RNAseq DESeq2 method.

[0050] As used herein, “asporin” or “ASPN” refer to NCBI Gene ID: 54829. This gene encodes a cartilage extracellular protein that is member of the small leucine-rich proteoglycan family. The encoded protein may regulate chondrogenesis by inhibiting transforming growth factor-beta 1- induced gene expression in cartilage. This protein also binds collagen and calcium and may induce collagen mineralization. Polymorphisms in the aspartic acid repeat region of this gene are associated with a susceptibility to osteoarthritis, and also with intervertebral disc disease. Alternative splicing of this gene results in multiple transcript variants. There are two identified isoforms of the protein - one with 379 amino acids (NCBI Reference Sequence: NP_060150.4) and one with 242 amino acids (NCBI Reference Sequence: NP_001180264.1).

[0051] As used herein, “collagen, type I, alpha 1” or “COL1A1” refers to the gene encoding pro- alpha2 chain of type I collagen NCBI Gene ID: 1277.

[0052] As used herein, “collagen type I, alpha 2” or “C0L1A2’ refers to the gene encoding a major protein component of type I collagen NCBI Gene ID: 1278.

[0053] As used herein “peroxisome proliferator-activated receptor gamma coactivator 1 -alpha (PGC-1a)” or “PPARGC1A” refers to NCBI Gene ID: 10891. This gene encodes the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a) protein. PGC-1a is a transcriptional coactivator that regulates genes involved in energy metabolism.

[0054] As used herein, “basic fibroblast growth factor” or “bFGF” refers to a growth factor that functions in angiogenesis, wound healing, tissue repair, learning and memory, and the morphogenesis of heart, bone, and brain. It is upregulated in response to inflammatory stimuli and in many tumors. bFGF is a common cell culture additive.

[0055] One embodiment described herein is a fabricated cell sheet composition, the composition comprising: a population of juvenile cartilage-derived chondrocytes (JCCs) with low gene expression of asporin (ASPN). In one aspect, the low gene expression of ASPN is below about 100 units as measured by an RNAseq DESeq2 method. In another aspect, the population of JCCs further comprises low gene expression of COL1A 1, COL1A2, or a combination thereof. In another aspect, the low gene expression of the sum of COL1A1 and COL1A2 is below about 10 x 105units as measured by an RNAseq DESeq2 method. In another aspect, the JCCs are derived from a human polydactyly resection surgery. In another aspect, the JCCs are a mixture of two or more different cell compositions.

[0056] Another embodiment described herein is a method of maintaining low gene expression of asporin (ASPN) in subcultured juvenile cartilage-derived chondrocytes (JCCs) for formation of cell sheets, the method comprising: treating the subcultured JCCs one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby maintaining low gene expression of ASPN in the subcultured JCCs for formation of cell sheets. In one aspect, the subcultured JCCs are continuously treated with the culture medium comprising bFGF. In another aspect, the subcultured JCCs are treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL. In another aspect, the method further comprises treating the subcultured JCCs with an additional agent to maintain low gene expression of ASPN.

[0057] Another embodiment described herein is a method for producing a cell sheet composition comprising chondrocytes, the method comprising: (a) collecting living cartilage tissue by scalpel under sterile conditions, wherein the cartilage tissue comprises JCCs; (b) cutting the collected cartilage tissue with scalpels into pieces; (c) collecting JCCs from the cartilage tissue through enzymatic treatment of the pieces of cartilage tissue and sub-culturing; and (d) culturing a mixture of JCCs in culture solution on a temperature-responsive polymer which has been coated onto a substrate surface of a cell culture support, wherein the temperature-responsive polymer has a lower critical solution temperature in water of 0-80 °C; (e) adjusting the temperature of the culture solution to below the lower critical solution temperature, whereby the substrate surface is made hydrophilic and adhesion of the cell sheet to the surface is weakened; and (f) detaching the cell sheet composition from the cell culture support, thereby producing a cell sheet composition.

[0058] Another embodiment described herein is a fabricated cell sheet composition having improved cartilage repair and regenerative efficacy, the composition comprising: a population of juvenile cartilage-derived chondrocytes (JCCs) comprising low gene expression of asporin (ASPN). In one aspect, the low gene expression of ASPN is below about 100 units as measured by an RNAseq DESeq2 method. In another aspect, the population of JCCs further comprises low gene expression of COL1A1, COL1A2, or a combination thereof. In another aspect, the low gene expression of the sum of COL1A1 and COL1A2 is below about 10 x 105units as measured by an RNAseq DESeq2 method. In another aspect, the composition generates an in vivo regenerative O’Driscoll (OD) score of greater than or equal to about 22 when implanted in vivo. In another aspect, the JCCs are derived from a human donor of 3 months to 16 years of age. In another aspect, the JCCs are derived from a human polydactyly resection surgery. In another aspect, the JCCs are a mixture of two or more different cell compositions. In another aspect, the JCCs are derived from an equine (horse) donor of less than about 2 years of age.

[0059] Another embodiment described herein is a method of maintaining low gene expression of asporin (ASPN) in subcultured juvenile cartilage-derived chondrocytes (JCCs) for formation of cell sheets having improved cartilage repair and regenerative efficacy, the method comprising: treating the subcultured JCCs one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby maintaining low gene expression of ASPN in the subcultured JCCs for formation of cell sheets. In another aspect, the subcultured JCCs are continuously treated with the culture medium comprising bFGF. In another aspect, the subcultured JCCs are treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL. In another aspect, low gene expression of ASPN is maintained in the subcultured JCCs up to at least 6 passages. In another aspect, the method further comprises treating the subcultured JCCs with an additional agent to maintain low gene expression of ASPN. In another aspect, the additional agent comprises an ASPN siRNA, an ASPN shRNA, an ASPN inhibitor, an agent that suppresses ASPN gene expression, or combinations thereof. In another aspect, the agent that suppresses ASPN gene expression comprises a PPARGIa inducer.

[0060] Another embodiment described herein is a method of enhancing the expansion of subcultured juvenile cartilage-derived chondrocytes (JCCs) for formation of cell sheets having improved cartilage repair and regenerative efficacy, the method comprising: treating the subcultured JCCs one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby enhancing the expansion of the subcultured JCCs for formation of cell sheets. In one aspect, the subcultured JCCs are continuously treated with the culture medium comprising bFGF. In another aspect, the subcultured JCCs are treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL.

[0061] Another embodiment described herein is a method of maintaining low gene expression of asporin (ASPN) in a culture of fabricated cell sheets comprising a population of juvenile cartilage- derived chondrocytes (JCCs) comprising low gene expression of ASPN and having improved cartilage repair and regenerative efficacy, the method comprising: treating the culture of fabricated cell sheets one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby maintaining low gene expression of ASPN in the culture of fabricated cell sheets. In another aspect, the culture of fabricated cell sheets is continuously treated with the culture medium comprising bFGF. In another aspect, the culture of fabricated cell sheets is treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL.

[0062] Another embodiment described herein is a method for producing a cell sheet composition comprising chondrocytes, the method comprising: (a) culturing a mixture of JCCs in culture solution on a temperature-responsive polymer which has been coated onto a substrate surface of a cell culture support, wherein the temperature-responsive polymer has a lower critical solution temperature in water of 0-80 °C; (b) adjusting the temperature of the culture solution to below the lower critical solution temperature, whereby the substrate surface is made hydrophilic and adhesion of the cell sheet to the surface is weakened; (c) detaching the cell sheet composition from the cell culture support, thereby producing a cell sheet composition, optionally wherein the method further comprises: (d) collecting cartilage tissue by scalpel under sterile conditions, wherein the cartilage tissue comprises JCCs; (e) cutting the collected cartilage tissue with scalpels into pieces; and (f) collecting JCCs from the cartilage tissue through enzymatic treatment of the pieces of cartilage tissue.

[0063] Another embodiment described herein is a method for repairing and / or regenerating cartilage in a subject in need thereof, the method comprising: treating the subject with a fabricated cell sheet composition comprising a population of juvenile cartilage-derived chondrocytes (JCCs) comprising low gene expression of asporin (ASPN). In another aspect, the population of JCCs comprising low gene expression of ASPN is pre-selected from a plurality of donor cell groups having different levels of ASPN gene expression. In another aspect, the subject is a human subject or an equine (horse) subject.

[0064] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0065] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0066] Clause 1. A fabricated cell sheet composition, the composition comprising: a population of juvenile cartilage-derived chondrocytes (JCCs) with low gene expression of asporin (ASPN).

[0067] Clause 2. The composition of clause 1 , wherein the low gene expression of ASPN is below about 100 units as measured by an RNAseq DESeq2 method.

[0068] Clause 3. The composition of clause 1 or 2, wherein the population of JCCs further comprises low gene expression of COL1A1, COL1A2, or a combination thereof.

[0069] Clause 4. The composition of any one of clauses 1-3, wherein the low gene expression of the sum of COL1A1 and COL1A2 is below about 10 * 105units as measured by an RNAseq DESeq2 method.

[0070] Clause 5. The composition of any one of clauses 1-4, wherein the JCCs are derived from a human polydactyly resection surgery.

[0071] Clause 6. The composition of any one of clauses 1-5, wherein the JCCs are a mixture of two or more different cell compositions.

[0072] Clause 7. A method of maintaining low gene expression of asporin (ASPN) in subcultured juvenile cartilage-derived chondrocytes (JCCs) for formation of cell sheets, the method comprising: treating the subcultured JCCs one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby maintaining low gene expression of ASPN in the subcultured JCCs for formation of cell sheets.

[0073] Clause 8. The method of clause 7, wherein the subcultured JCCs are continuously treated with the culture medium comprising bFGF.

[0074] Clause 9. The method of clause 7 or 8, wherein the subcultured JCCs are treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL.

[0075] Clause 10. The method of any one of clauses 7-9, further comprising treating the subcultured JCCs with an additional agent to maintain low gene expression of ASPN.

[0076] Clause 11. A method for producing a cell sheet composition comprising chondrocytes, the method comprising:

[0077] (a) collecting living cartilage tissue by scalpel under sterile conditions, wherein the cartilage tissue comprises JCCs;

[0078] (b) cutting the collected cartilage tissue with scalpels into pieces;

[0079] (c) collecting JCCs from the cartilage tissue through enzymatic treatment of the pieces of cartilage tissue and sub-culturing;

[0080] (d) culturing a mixture of JCCs in culture solution on a temperature-responsive polymer which has been coated onto a substrate surface of a cell culture support, wherein the temperature-responsive polymer has a lower critical solution temperature in water of 0-80 °C;

[0081] (e) adjusting the temperature of the culture solution to below the lower critical solution temperature, whereby the substrate surface is made hydrophilic and adhesion of the cell sheet to the surface is weakened; and

[0082] (f) detaching the cell sheet composition from the cell culture support, thereby producing a cell sheet composition.

[0083] Clause 12. A fabricated cell sheet composition having improved cartilage repair and regenerative efficacy, the composition comprising: a population of juvenile cartilage- derived chondrocytes (JCCs) comprising low gene expression of asporin (ASPN).

[0084] Clause 13. The composition of clause 12, wherein the low gene expression of ASPN is below about 100 units as measured by an RNAseq DESeq2 method.

[0085] Clause 14. The composition of clause 12 or 13, wherein the population of JCCs further comprises low gene expression of COL1A1, COL1A2, or a combination thereof. Clause 15. The composition of any one of clauses 12-14, wherein the low gene expression of the sum of COL1A1 and COL1A2 is below about 10 x 105units as measured by an RNAseq DESeq2 method.

[0086] Clause 16. The composition of any one of clauses 12-15, wherein the composition generates an in vivo regenerative O’Driscoll (OD) score of greater than or equal to about 22 when implanted in vivo.

[0087] Clause 17. The composition of any one of clauses 12-16, wherein the JCCs are derived from a human donor of 3 months to 16 years of age.

[0088] Clause 18. The composition of any one of clauses 12-17, wherein the JCCs are derived from a human polydactyly resection surgery.

[0089] Clause 19. The composition of any one of clauses 12-18, wherein the JCCs are a mixture of two or more different cell compositions.

[0090] Clause 20. The composition of any one of clauses 12-19, wherein the JCCs are derived from an equine (horse) donor of less than about 2 years of age.

[0091] Clause 21. A method of maintaining low gene expression of asporin (ASPN) in subcultured juvenile cartilage-derived chondrocytes (JCCs) for formation of cell sheets having improved cartilage repair and regenerative efficacy, the method comprising: treating the subcultured JCCs one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby maintaining low gene expression of ASPN in the subcultured JCCs for formation of cell sheets.

[0092] Clause 22. The method of clause 21 , wherein the subcultured JCCs are continuously treated with the culture medium comprising bFGF.

[0093] Clause 23. The method of clause 21 or 22, wherein the subcultured JCCs are treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL.

[0094] Clause 24. The method of any one of clauses 21-23, wherein low gene expression of ASPN is maintained in the subcultured JCCs up to at least 6 passages.

[0095] Clause 25. The method of any one of clauses 21-24, further comprising treating the subcultured JCCs with an additional agent to maintain low gene expression of ASPN.

[0096] Clause 26. The method of clause 25, wherein the additional agent comprises an ASPN siRNA, an ASPN shRNA, an ASPN inhibitor, an agent that suppresses ASPN gene expression, or combinations thereof.

[0097] Clause 27. The method of clause 26, wherein the agent that suppresses ASPN gene expression comprises a PPARGIa inducer. Clause 28. A method of enhancing the expansion of subcultured juvenile cartilage-derived chondrocytes (JCCs) for formation of cell sheets having improved cartilage repair and regenerative efficacy, the method comprising: treating the subcultured JCCs one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby enhancing the expansion of the subcultured JCCs for formation of cell sheets.

[0098] Clause 29. The method of clause 28, wherein the subcultured JCCs are continuously treated with the culture medium comprising bFGF.

[0099] Clause 30. The method of clause 28 or 29, wherein the subcultured JCCs are treated with a concentration of bFGF ranging from about 0.5 ng / mL to 10 about ng / mL.

[0100] Clause 31. A method of maintaining low gene expression of asporin (ASPN) in a culture of fabricated cell sheets comprising a population of juvenile cartilage-derived chondrocytes (JCCs) comprising low gene expression of ASPN and having improved cartilage repair and regenerative efficacy, the method comprising: treating the culture of fabricated cell sheets one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby maintaining low gene expression of ASPN in the culture of fabricated cell sheets.

[0101] Clause 32. The method of clause 31 , wherein the culture of fabricated cell sheets is continuously treated with the culture medium comprising bFGF.

[0102] Clause 33. The method of clause 31 or 32, wherein the culture of fabricated cell sheets is treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL.

[0103] Clause 34. A method for producing a cell sheet composition comprising chondrocytes, the method comprising:

[0104] (a) culturing a mixture of JCCs in culture solution on a temperature-responsive polymer which has been coated onto a substrate surface of a cell culture support, wherein the temperature-responsive polymer has a lower critical solution temperature in water of 0-80 °C;

[0105] (b) adjusting the temperature of the culture solution to below the lower critical solution temperature, whereby the substrate surface is made hydrophilic and adhesion of the cell sheet to the surface is weakened;

[0106] (c) detaching the cell sheet composition from the cell culture support, thereby producing a cell sheet composition, optionally wherein the method further comprises (d) collecting cartilage tissue by scalpel under sterile conditions, wherein the cartilage tissue comprises JCCs;

[0107] (e) cutting the collected cartilage tissue with scalpels into pieces; and

[0108] (f) collecting JCCs from the cartilage tissue through enzymatic treatment of the pieces of cartilage tissue.

[0109] Clause 35. A method for repairing and / or regenerating cartilage in a subject in need thereof, the method comprising: treating the subject with a fabricated cell sheet composition comprising a population of juvenile cartilage-derived chondrocytes (JCCs) comprising low gene expression of asporin (ASPN).

[0110] Clause 36. The method of clause 35, wherein the population of JCCs comprising low gene expression of ASPN is pre-selected from a plurality of donor cell groups having different levels of ASPN gene expression.

[0111] Clause 37. The method of clause 35 or 36, wherein the subject is a human subject or an equine (horse) subject.

[0112] EXAMPLES

[0113] Cartilage Sourcing

[0114] Primary human cartilage cells were isolated from 8 amputated polydactylous fingers and a toe from 8 juvenile patients aged between 6 and 30 months (5 males, 3 females; 14.6 ± 6.9 months; Caucasian). These patients underwent routine polydactyly surgery, and de-identified tissue discards from these surgeries were collected at Intermountain Primary Children’s Hospital (Salt Lake City, USA). Institutional Review Board oversight from the University of Utah (USA) and Intermountain Primary Children’s Hospital was waived due to the use of de-identified routine surgical discards for research purposes.

[0115] Fabrication of Juvenile Chondrocyte Sheets

[0116] Cartilage tissues from polydactyly surgical discards were dissected, enzymatically digested, and cultured for cryogenic cell banking, and JCC sheets were fabricated according to methods described by Kondo et al., Regen. Med. 6(1): 1-11 (2021). Briefly, human cartilage tissue was cut into <4 mm2pieces by scalpel and incubated with 5 mg / mL Type 1 collagenase (LS004197, Worthington Biochemical, Lakewood, USA) at 37 °C for 1.5-3.0 h. Resulting cells were filtered through a 100-pm cell strainer, washed with saline, and then resuspended in chondrocyte culture medium (DMEM-F12, 11320082, Thermo Fisher Scientific, Waltham, USA) containing 1% antibiotic-antimycotic (15240062, Thermo Fisher) and 20% fetal bovine serum (FBS) (16000044, Thermo Fisher). Isolated chondrocytes were seeded at 5,000-10,000 cells / cm2on polystyrene dishes (CELLTREAT, Pepperell, USA) in chondrocyte culture medium. At the first medium change on day 4, 100 pg / mL L-ascorbic acid phosphate magnesium salt n- hydrate (013-19641 , Fujifilm Wako Pure Chemical, Osaka, Japan) was added and the medium was replaced. Cells were passaged with this medium thereafter. Sub-confluent cells were suspended in STEM-CELLBANKER GMP grade media (Zenoaq, Fukushima, Japan) and cryopreserved at the end of P0. Serial subculture was performed with thawed cells at an initial density of 10,000 cells / cm2passaged every 3-5 days. To create passage 2 (P2) JCC sheets, P1 thawed cryopreserved cells were seeded and sub-confluent P1 cells were collected, then seeded at a density of 10,000 cells / cm2on 23-mm diameter temperature-responsive cell culture inserts (SSCW Insert, Cell Sheet Tissue Engineering Regenerative Medicine Initiative, CSTERMi, Tokyo, Japan). Cell culture medium was changed every 2-4 days. After 2 weeks of culture, confluent cell sheets were harvested with forceps after incubation at room temperature. P2 JCC sheets from all human donors were used for in vivo transplantation as described below. In some experiments, basic fibroblast growth factor (bFGF) was used at 1.0 ng / mL at primary culture or after thawing the frozen vials. siRNA was purchased and optimal concentration was determined according to manufacturer’s protocol (Horizon).

[0117] Nude Rat Cartilage Defect Model and Human Cell Sheet Transplantation

[0118] All in vivo implant procedures were approved by the Institutional Animal Care and Use Committee (IACUC, University of Utah). A total of 30 nude rats (6-week-old), both male and female, were purchased from Charles River Laboratories (Wilmington, USA). After a week of acclimatization at the animal facility, rats were randomized to each experimental condition by comparable body weights. Under anesthesia using isoflurane and O2gas, medial parapatellar incision was made on the right knee and the patella was laterally dislocated. A focal chondral defect (diameter 2 mm; depth 300-400 pm) was created on the patellofemoral groove using an electric grinder with minimal damage to the subchondral bone. Freshly prepared JCC sheets were washed with saline, then cut in half with a sterile razor blade, and single sheet halves were transplanted into the defect (FIG. 1 A). Sheets attach spontaneously without suturing to the defect surface. The patella was anatomically repositioned and the quadriceps femoris muscle and skin were sutured. All groups received surgery with comparable duration regardless of the treatment conditions. Only one implant was placed in one chondral defect per animal. Animals were sacrificed after 4-weeks for further histological evaluations. The employed animal numbers for cohorts were determined by a significance level of 5% and 80% power using pilot study results. Results from one rat were excluded due to defect site positioning outside of the desired rat patellofemoral groove anatomical area.

[0119] Harvested rat knee tissue was fixed in 4% paraformaldehyde for four days and decalcified in RapidCal Immuno (BBC Biochemical, Mount Vernon, USA) for one day at RT. Samples were embedded in paraffin blocks and then cut into 5-pm transverse sections with a microtome. P2 JCC cell sheets from donor 1 were implanted in 3 female rats, cell sheets from donor 2 into 2 male and 1 female rat, cell sheets from donor 3 into 3 female rats, cell sheets from donor 4 into 3 male rats, cell sheets from donor 5 into 3 male and 3 female rats each, and cell sheets from donor 6, 7, and 8 into 3 male rats. Defect models served as controls using 3 male rats.

[0120] Histological Evaluation of Harvested Cartilage

[0121] Allocation and assessment were done by multiple investigators for randomization. Histological samples from each rat defect site were prepared separately for Safranin O, collagen type 1 (COL1), collagen type 2 (COL2), and human-specific vimentin (hVIM) staining. Slides were deparaffinized by baking in an oven at 65 °C and subsequent washes with xylene and ethanol. Safranin O staining was conducted according to standard methods. Briefly, samples were stained for 5 min with Weigert’s Iron Hematoxylin (MilliporeSigma), 5 min with 0.5 g / L Fast Green (MilliporeSigma), and 5 min with 0.1 % Safranin O (MilliporeSigma). After Safranin O staining, slides were randomized and scored with modified O’Driscoll scoring and the observer was blinded regarding JCC donor information.

[0122] For immunostaining, histology samples were rehydrated and protease K (S3020, Agilent Technologies, Santa Clara, USA) was used for antigen retrieval for COL2 staining. Peroxidase blocking was performed with 3% hydrogen peroxide (216763, MilliporeSigma). After blocking with 5% donkey serum and 0.1% Triton-X 100 in PBS for 1 h, samples were treated with primary antibodies to COL1 , COL2, and hVIM at 4 °C overnight. Polyclonal goat anti-COL1 (1 :200, SouthernBiotech, Birmingham, USA), monoclonal mouse anti-COL2 (1:200, 2B1.5, Thermo Fisher, USA), and monoclonal rabbit anti-hVIM (1 :200, SP20, Abeam) were used as primary antibodies. Normal goat IgG (1 :50, NI02, MilliporeSigma), normal mouse lgG2a (1:100, X0943, Agilent), or normal rabbit IgG (1 :1 ,500, X0903, Agilent) were used as isotype controls. Horseradish peroxidase (HRP)-conjugated donkey anti-goat antibody (1 :1 ,000, 705-035-147, Jackson ImmunoResearch, West Grove, USA) was used for type 1 collagen. HRP-conjugated goat anti-mouse antibody (1 :1 ,000, 115-035-166, Jackson) was used for type 2 collagen. HRP- conjugated goat anti-rabbit antibody (1 :1 ,000, 111-035-144, Jackson) was used for hVIM staining. ImmPACT DAB Peroxidase (HRP) Substrate (SK-4105, Vector Laboratories, Burlingame, USA) was used as a chromogen. Brightfield images were taken with a BX41 microscope and processed with AmScope Software. For quantitative IHC, the positive cell area across the entire cell sheet was measured and expressed as percent using Imaged software (NIH) (%COL1 and %COL2 areas, FIG. 8). Safranin O staining and hVIM staining of the serial sections were used as a reference. Ratios of COL1 to COL2 in both the total defect and regenerating areas (COL2 / 1 ratio) were also calculated.

[0123] Correlation coefficients for each modified O’Driscoll score and %COL1 area, %COL2 area, and COL2 / 1 ratio were calculated. For donors treated with P2 JCC sheets, scores were compared between donors and the defect-only group and divided into “more effective” and “less effective” groups based on average scores. Significant differences between each group and the defect- only group in each category of modified O’Driscoll score were evaluated. A summary of the methodological process is described in FIG. 1A.

[0124] Selection of Efficacious Cell Sheet in In Vivo Model

[0125] Results from repaired rodent cartilage defects were divided to two groups using modified O’Driscoll scoring performed by multiple blinded examiners. See Table 1. Knee histological samples with the score >21.0 were considered as “efficacious”. Therefore, the donors that were used in the group was defined as “more effective donors.” The other group with the score below 21.0 was defined as “less effective donors,” which still showed better scores than defect-only control group: score: 15.8 ± 1.3.

[0126] Table 1. Modified O’Driscoll Score

[0127] I. Nature of Predominant

[0128] Hyaline cartilage 4

[0129] Mostly hyaline cartilage 3

[0130] Mixed hyaline and fibrocartilage 2

[0131] Mostly fibrocartilage 1

[0132] Some fibrocartilage, mostly non-chondrocytic cells 0

[0133] II. Structural Characteristics

[0134] A. Surface regularity

[0135] Smooth and intact 3

[0136] Superficial horizontal lamination 2

[0137] Fissures 1

[0138] Severe disruption, including fibrillation 0

[0139] B. Structural integrity, homogeneity

[0140] Normal 2 Slight disruption 1

[0141] Severe disintegration, disruptions 0

[0142] C. Thickness

[0143] 100% of adjacent cartilage 2

[0144] 50-100% of normal cartilage 1

[0145] 0-50% of normal cartilage 0

[0146] D. Bonding to adjacent cartilage

[0147] Bonded at both ends of graft 2

[0148] Bonded at one end or partially at both ends 1

[0149] Not bonded 0

[0150] III. Freedom from Cellular Changes of Degeneration

[0151] A. Hypocellularity

[0152] Normal cellularity 2

[0153] Slight hypocellularity 1

[0154] Moderate hypocellularity or hypercellularity 0

[0155] B. Chondrocyte clustering

[0156] No clusters 2

[0157] <25% of the cells 1

[0158] 25-100% of the cells 0

[0159] IV. Freedom from degenerative changes in adjacent cartilage

[0160] Normal cellularity, no clusters, normal staining 3

[0161] Normal cellularity, mild clusters, moderate staining 2

[0162] Mild or moderate hypo / hypercellularity, slight staining 1

[0163] Sever hypocellularity, poor or no staining 0

[0164] V. Subchondral bone

[0165] A. Reconstruction of subchondral bone

[0166] Normal 3

[0167] Reduced subchondral bone reconstruction 2

[0168] Minimal subchondral bone reconstruction 1

[0169] No subchondral bone reconstruction 0

[0170] B. Inflammatory response in subchondral bone region

[0171] None / mild 2

[0172] Moderate 1

[0173] Severe 0

[0174] VI. Safranin O staining

[0175] Normal or near normal 3

[0176] Moderate 2

[0177] Slight 1

[0178] None 0

[0179] Total maximum score: 28 Statistical Analysis

[0180] Numerical results are expressed as a mean and standard deviation. Pearson’s correlation coefficient was used to identify significant relationships between modified O’Driscoll scores. ANOVA with post-hoc Tukey-Kramer testing was used to identify significant differences between donors in each score and between groups in each category of modified O’Driscoll score. All tests were performed at a significance level of p < 0.05. Statistical analysis was performed with statistical package R (version 4.2.0).

[0181] RNA-seq and Analysis

[0182] RNA was extracted from harvested cell sheets using RNeasy mini kit (Qiagen, Hilden, Germany). After RNA integrity and quantity was checked, a cDNA library was constructed and used for pair-end sequencing using NovaSeq 6000 (Illumina, San Diego, CA, USA). Sequencing data were uploaded to the Galaxy web platform, and a public server at usegalaxy.org was used to analyze the data. After trimming of Illumina-specific sequences using Trimmomatic version 0.38, the sequence reads were aligned to the human genome reference sequence (hg38) using STAR version 2.7.5b. Gene-level assignment was performed using featureCounts version 1.6.4. Acquired data quality was checked using MultiQC and confirmed to be high. The gene expression matrix with raw gene counts was used for differential gene expression analysis using DESeq2 version 1.22.1 , which includes size factor normalization. Clustering analysis was performed with differentially expressed 1 ,554 genes of Padj <0.05, fold change > Abs(2). Gene ontology analysis was performed using the Metascape platform at metascape.org with the differentially expressed gene list of Padj < 0.05, fold change >2 or fold change <-2. Volcano plot and heatmap were also created by using galaxy tools based on the thresholds mentioned above.

[0183] Comparison of Gene Expression Rank between New and Past Samples

[0184] 1. Normalization and Expression Extraction:

[0185] Normalize RNA-Seq data using DESeq2 (e.g., vst ( ) or rlog O ).

[0186] Extract expression values for the gene of interest (e.g., “GeneA”) from both past and new samples. vsd <- vst (dds , blind=FALSE) gene expressi on <- assay (vsd) [ "GeneA" , ]

[0187] 2. Rank Comparison:

[0188] Rank the expression values of the gene in past samples. Compare the rank of the new sample within the past sample distribution. past_sampl es <- assay (vsd) [ "GeneA" , past_sampl e_indi ces] new sample <- assay (vsd) [ "GeneA" , new sample index] past ranks <- rank (pas t samples) new_sample_rank <- rank (c (past_samples , new_sample) )

[0189] 3. Visualization:

[0190] Visualize gene expression distribution using heatmap or boxplots for comparison. phea tmap (assay (vsd) [c ( "GeneA" , "GeneB" , "GeneC") , ] ) boxplot (past sampl es , new sample , names=c ("Past Sampl es", "New Sample") )

[0191] Assessment of in vivo cartilage defect regeneration capacity is important to improve cartilage regenerative medicine products. Here, a nude rat knee cartilage defect model was employed as a platform to implant and test human juvenile chondrocyte sheets. JCC-regenerated histological samples were confirmed positive for hVIM immunostaining, supporting that new cartilage originates from implanted JCC human cells (FIG. 7). Representative histological samples are shown in FIG. 5B.

[0192] Development of improved, validated and scalable cartilage regeneration strategies remains an urgent clinical challenge. Assessment of reliable human cell-derived regenerative approaches is integral to assuring clinical progress. The potential for living human juvenile cartilage as an allogeneic cell source for regenerating cartilage is long recognized. Although multiple histological scoring systems have been introduced as part of regenerative outcomes analysis, correlations of these systems to human cell-based approaches are not fully investigated. Modified O’Driscoll scoring is widely used to assess in vivo cartilage repair in defect models and has been reported to be superior to other scores in terms of coefficient of repeatability. This study showed variations in O’Driscoll scores, %COL1- and %COL2-positive stained defect histological section areas, and COL2 / 1 ratios for human JCC sheets derived from various donors in an established nude rat defect model. Modified O’Driscoll scores correlated negatively with %COL1 areas and positively with %COL2 areas and COL2 / 1 ratios, linking the histological O’Driscoll cartilage scoring criteria to recognized cartilage-specific collagen matrix deposition. A previous in vivo study of particulated juvenile allograft cartilage in porcine articular cartilage defects showed that regenerated hyaline cartilage was found at only 20.1 % abundance 4 weeks after graft transplantation. Clinically, ACI outcomes report only 15% of patients exhibiting type 2 collagen- dominant cartilage. Thus, clinical regeneration of hyaline cartilage is difficult and unreliable with currently available cell therapies. In contrast, the in vivo data with human juvenile-sourced cell sheet treatment showed >80% COL2 and ~10% COL1 stained areas after 4 weeks of treatment with “more effective” donors in this model (FIG. 2C), suggesting high cartilage regenerative potential in defects using JCC sheets. Results also highlighted the variations in chondrogenic potential of JCC sheets from different donors (FIG. 2C; Table 2). A single juvenile polydactyly donor can theoretically produce 2-3 x io3JCC sheets at P2. To leverage this JCC scalability across allogenic donors, a reliable assessment system as proposed in this study is important for human donor cell screening that reliably delineates more effective donor cells from less effective donor cells.

[0193] Based on the scoring shown on histological analysis of rodent cartilage defects treated with JCC sheets, juvenile JCC donors could be divided into “more effective” and “less effective” groups (FIG. 2C). Between these groups, significant differences in donors were observed in scoring categories “Nature of predominant tissue,” “Reconstruction of subchondral bone,” and “Safranin O staining.” The significant difference in “Reconstruction of subchondral bone” scoring among JCC sheet donor sheets is noteworthy. Subchondral bone absorbs most of the applied joint mechanical force and provides essential mechanical support for overlying articular cartilage; it may be an essential component for cartilage regeneration. The importance of the union between the graft and subchondral bone in a rabbit model of costal osteochondral grafts has been reported, showing histological union between the graft and the subchondral bone at 6 weeks after transplantation. A dynamic relationship between articular cartilage and subchondral bone has been reported and suggested that abnormality in either could disturb the homeostatic balance of the bone / cartilage unit. Hence, lack of subchondral bone repair / reconstruction potential might undermine cartilage regeneration in the “less effective” group.

[0194] Safranin O staining showed structures that appear to be blood vessels in the subchondral layer in the “less effective” group at the 4-week time point, not observed in the “more effective” group at 4 weeks (FIG. 8A-B). Angiogenesis is a necessary process for bone remodeling, and its presence and staging in subchondral bone in joints with OA is recognized. Thus, subchondral bone remodeling and associated neovascular dynamics post-injury may be completed earlier in the “more effective” group than in the “less effective” group as part of defect repair. While details to describe the defect healing process within 4 weeks remain tentative, at 2 weeks postimplantation with a “more effective donor,” blood vessels were found in the subchondral bone and subchondral bone remodeling is not yet completed (FIG. 8C).

[0195] The scoring category “Structural characteristics” produced high scores in sheet- transplanted groups. A smooth regenerated tissue surface is required for low-friction articulation. Implant integration with surrounding cartilage is critical for successful cartilage repair to enable normal stress distribution with weight-bearing and prevent tissue degeneration. Thus, “Structural characteristics” is also an essential factor for reliable hyaline cartilage regeneration. Cell sheet implantation exhibited spontaneous sheet integration at the tissue site without suturing, producing the observed rapid hyaline cartilage structural regeneration reflected in the high scores in this category.

[0196] Analogous allogenic cell sheet transplantation studies can be performed in outbred animals, but cell properties, especially chondrogenic potential, are highly variable among different animal species. Hence, predictive outcomes extrapolated to human potential will be difficult and speculative without direct correlations. The athymic rat model employed here enables safety and efficacy testing of human cell products in the in vivo knee synovial articulating joint environment. A rabbit xenotransplantation model using immunosuppressing drugs is also reported for this purpose, although acute pharmacological immuno-suppression could affect natural healing cascades. Future studies should consider intrinsic limitations of these xenogeneic models: biomechanical loading of the articulating surfaces and its influences on cartilage healing are distinct between species, healing processes are likely distinct from that of allogeneic transplants, and essential involvement of all host immune cells in cartilage repair is lacking in immune- incompetent models. Nonetheless, it is believed that the nude rat cartilage implant model employed here provides useful mechanistic and potency screening of human cell transplantation products for cartilage regeneration. Host immune response to allogeneic JCC sheet transplantation in inflamed and post-surgery conditions to validate engraftment, safety, and efficacy of this potent human juvenile cell-based therapy is warranted.

[0197] As these donor screening results rely on JCC sheet transplantation in animal cartilage defects, a time- and money-intensive method, a different screening method to predict JCC donor efficacy based on analysis of banked donor cells is desirable. JCC donor cells used in rodent studies above (classified as “more effective” and less effective”) were further analyzed for specific molecular markers that correlated strongly with either “more” or “less” effective classifications found in vivo in rodent repair.

[0198] This study establishes a combined histological assessment system for human cell products for cartilage repair in a nude rat knee cartilage defect model. The system stratifies regenerative effects of human juvenile cartilage-derived chondrocyte (JCC) sheets derived from various juvenile donors. Scoring differences identified in histological evaluations of various juvenile JCC donor cartilage repair potentials are attributed to Safranin O staining and subchondral bone remodeling. Most JCC sheet implant outcomes exhibit excellent “Structural characteristics” scoring in this model. The model outcomes extend and support previous work, demonstrating the benefits of human juvenile chondrocyte sourcing to yield potent cell sheet constructs that spontaneously adhere to chondral defects, and rapidly integrate to regenerate full thickness hyaline-like cartilage. While allogeneic transplantation studies are vital for evaluating immune response and rejection, leveraging this implant regenerative model to screen human cell sources for critical quality attributes is essential for product development. This process enables reliable cartilage regeneration and facilitates donor selection, banking, scaling, and achieving desired cost-effectiveness for allogeneic living cartilage implants.

[0199]

[0200]

Claims

CLAIMSWhat is claimed:

1. A fabricated cell sheet composition, the composition comprising: a population of juvenile cartilage-derived chondrocytes (JCCs) with low gene expression of asporin (ASPN).

2. The composition of claim 1 , wherein the low gene expression of ASPN is below about 100 units as measured by an RNAseq DESeq2 method.

3. The composition of claim 1 , wherein the population of JCCs further comprises low gene expression of COL1A1, COL1A2, or a combination thereof.

4. The composition of claim 1 , wherein the low gene expression of the sum of COL1A1 and COL1A2 is below about 10 x 105units as measured by an RNAseq DESeq2 method.

5. The composition of claim 1 , wherein the JCCs are derived from a human polydactyly resection surgery.

6. The composition of claim 1 , wherein the JCCs are a mixture of two or more different cell compositions.

7. A method of maintaining low gene expression of asporin (ASPN) in subcultured juvenile cartilage-derived chondrocytes (JCCs) forformation of cell sheets, the method comprising: treating the subcultured JCCs one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby maintaining low gene expression of ASPN in the subcultured JCCs for formation of cell sheets.

8. The method of claim 7, wherein the subcultured JCCs are continuously treated with the culture medium comprising bFGF.

9. The method of claim 7, wherein the subcultured JCCs are treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL.

10. The method of claim 7, further comprising treating the subcultured JCCs with an additional agent to maintain low gene expression of ASPN.

11. A method for producing a cell sheet composition comprising chondrocytes, the method comprising:(a) collecting living cartilage tissue by scalpel under sterile conditions, wherein the cartilage tissue comprises JCCs;(b) cutting the collected cartilage tissue with scalpels into pieces;(c) collecting JCCs from the cartilage tissue through enzymatic treatment of the pieces of cartilage tissue and sub-culturing;(d) culturing a mixture of JCCs in culture solution on a temperature-responsive polymer which has been coated onto a substrate surface of a cell culture support, wherein the temperature-responsive polymer has a lower critical solution temperature in water of 0-80 °C;(e) adjusting the temperature of the culture solution to below the lower critical solution temperature, whereby the substrate surface is made hydrophilic and adhesion of the cell sheet to the surface is weakened; and(f) detaching the cell sheet composition from the cell culture support, thereby producing a cell sheet composition.

12. A fabricated cell sheet composition having improved cartilage repair and regenerative efficacy, the composition comprising: a population of juvenile cartilage-derived chondrocytes (JCCs) comprising low gene expression of asporin (ASPN).

13. The composition of claim 12, wherein the low gene expression of ASPN is below about 100 units as measured by an RNAseq DESeq2 method.

14. The composition of claim 12, wherein the population of JCCs further comprises low gene expression of COL1A1, COL1A2, or a combination thereof.

15. The composition of claim 12, wherein the low gene expression of the sum of COL1A1 and COL1A2 is below about 10 x 105units as measured by an RNAseq DESeq2 method.

16. The composition of claim 12, wherein the composition generates an in vivo regenerative O’Driscoll (OD) score of greater than or equal to about 22 when implanted in vivo.

17. The composition of claim 12, wherein the JCCs are derived from a human donor of 3 months to 16 years of age.

18. The composition of claim 12, wherein the JCCs are derived from a human polydactyly resection surgery.

19. The composition of claim 12, wherein the JCCs are a mixture of two or more different cell compositions.

20. The composition of claim 12, wherein the JCCs are derived from an equine (horse) donor of less than about 2 years of age.

21. A method of maintaining low gene expression of asporin (ASPN) in subcultured juvenile cartilage-derived chondrocytes (JCCs) for formation of cell sheets having improved cartilage repair and regenerative efficacy, the method comprising: treating the subcultured JCCs one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby maintaining low gene expression of ASPN in the subcultured JCCs for formation of cell sheets.

22. The method of claim 21 , wherein the subcultured JCCs are continuously treated with the culture medium comprising bFGF.

23. The method of claim 21 , wherein the subcultured JCCs are treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL.

24. The method of claim 21 , wherein low gene expression of ASPN is maintained in the subcultured JCCs up to at least 6 passages.

25. The method of claim 21 , further comprising treating the subcultured JCCs with an additional agent to maintain low gene expression of ASPN.

26. The method of claim 25, wherein the additional agent comprises an ASPN siRNA, an ASPN shRNA, an ASPN inhibitor, an agent that suppresses ASPN gene expression, or combinations thereof.

27. The method of claim 26, wherein the agent that suppresses ASPN gene expression comprises a PPARGIa inducer.

28. A method of enhancing the expansion of subcultured juvenile cartilage-derived chondrocytes (JCCs) for formation of cell sheets having improved cartilage repair and regenerative efficacy, the method comprising: treating the subcultured JCCs one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby enhancing the expansion of the subcultured JCCs for formation of cell sheets.

29. The method of claim 28, wherein the subcultured JCCs are continuously treated with the culture medium comprising bFGF.

30. The method of claim 28, wherein the subcultured JCCs are treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL.

31. A method of maintaining low gene expression of asporin (ASPN) in a culture of fabricated cell sheets comprising a population of juvenile cartilage-derived chondrocytes (JCCs) comprising low gene expression of ASPN and having improved cartilage repair and regenerative efficacy, the method comprising: treating the culture of fabricated cell sheets one or more times with a culture medium comprising basic fibroblast growth factor (bFGF), thereby maintaining low gene expression of ASPN in the culture of fabricated cell sheets.

32. The method of claim 31 , wherein the culture of fabricated cell sheets is continuously treated with the culture medium comprising bFGF.

33. The method of claim 31 , wherein the culture of fabricated cell sheets is treated with a concentration of bFGF ranging from about 0.5 ng / mL to about 10 ng / mL.

34. A method for producing a cell sheet composition comprising chondrocytes, the method comprising:(a) culturing a mixture of JCCs in culture solution on a temperature-responsive polymer which has been coated onto a substrate surface of a cell culture support, wherein the temperature-responsive polymer has a lower critical solution temperature in water of 0-80 °C;(b) adjusting the temperature of the culture solution to below the lower critical solution temperature, whereby the substrate surface is made hydrophilic and adhesion of the cell sheet to the surface is weakened;(c) detaching the cell sheet composition from the cell culture support, thereby producing a cell sheet composition, optionally wherein the method further comprises(d) collecting cartilage tissue by scalpel under sterile conditions, wherein the cartilage tissue comprises JCCs;(e) cutting the collected cartilage tissue with scalpels into pieces; and(f) collecting JCCs from the cartilage tissue through enzymatic treatment of the pieces of cartilage tissue.

35. A method for repairing and / or regenerating cartilage in a subject in need thereof, the method comprising: treating the subject with a fabricated cell sheet composition comprising a population of juvenile cartilage-derived chondrocytes (JCCs) comprising low gene expression of asporin (ASPN).

36. The method of claim 35, wherein the population of JCCs comprising low gene expression of ASPN is pre-selected from a plurality of donor cell groups having different levels of ASPN gene expression.

37. The method of claim 35, wherein the subject is a human subject or an equine (horse) subject.

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