Multi-tissue organoid products and methods

Abstract

The method of making multi-tissue organoid generally includes obtaining pluripotent stem cell (PSC), and inducing the growth of multi-tissue organoid by suspending and culturing the harvested PSC in culture vessel.Another method of making multi-tissue organoid generally includes introducing pluripotent stem cell into cell culture medium containing hyaluronic acid, transferring pluripotent stem cell into cell culture device that does not contain three-dimensional matrix, culturing pluripotent stem cell in cell culture device for at least 1 week, and making multi-tissue organoid that comprises cartilage, bone, fibrous connective tissue, brain tissue, or epithelial tissue, or combination thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 244,991, filed September 16, 2021, which is incorporated by reference in its entirety. Summary of the Invention

[0002] In one aspect, the present disclosure describes the method for producing multi-tissue organoid (MTO).Generally, the method includes obtaining pluripotent stem cell (PSC) and inducing the growth of multi-tissue organoid by suspending and culturing the harvested PSC in culture vessel.

[0003] In another aspect, the present disclosure describes the alternative method of making multi-tissue organoid (MTO).Generally, the method includes: introducing pluripotent stem cells into the cell culture medium containing hyaluronic acid, transferring pluripotent stem cells into the cell culture device that does not contain three-dimensional matrix, culturing pluripotent stem cells in the cell culture device for at least 1 week, and making the multi-tissue organoid that comprises cartilage, bone, fibrous connective tissue, brain tissue, or epithelial tissue, or combinations thereof.

[0004] In one or more embodiments of any of the methods, the cell culture medium comprises at least one of hyaluronic acid, fibroblast growth factor, transforming growth factor beta (TGFβ), growth differentiation factor 5 (GDF-5), DMEM / F12, magnesium L-ascorbic acid-2-phosphate, sodium selenium, insulin, NaHCO3, transferrin, TGFβ1, NODAL, or bone morphogenetic protein 2 (BMP-2).

[0005] In one or more embodiments of either method, the cell culture device includes a second cell culture medium.

[0006] In one or more embodiments of any of the methods, culturing the cells in the cell culture device includes culturing the cells at room temperature.

[0007] In one or more embodiments of any of the methods, culturing the cells in the cell culture device includes culturing the cells at 37°C.

[0008] In one or more embodiments of either method, the PSCs are incubated in a bioreactor.

[0009] In one or more embodiments of any of the methods, the organoids comprise cartilage, bone, fibrous connective tissue, or a combination thereof.

[0010] In one or more embodiments of either method, the organoid comprises neural cells or neural tissue.

[0011] In one or more embodiments of any of the methods, the method further comprises isolating the organoids.

[0012] In one or more embodiments of any method, the method further comprises disaggregating the cells of organoid to generate a population of individualized cells.In one or more of these embodiments, the disaggregated cells comprise chondrocytes.In one or more of these embodiments of any method, the method further comprises making chondrocytes form chondrospheres.

[0013] In one or more embodiments of any of the methods, the organoids comprise cells expressing transforming growth factor beta 1 (TGFβ1); cells expressing fibroblast growth factor 2 (FGF2); cells expressing bone morphogenetic protein 2 (BMP2); cells expressing bone morphogenetic protein 6 (BMP6); cells expressing growth differentiation factor 5 (GDF5); cells expressing secreted frizzled-related protein 1 (SFRP1); cells expressing inhibin subunit beta A (INHβA); cells expressing transforming growth factor beta 3 (TGFβ3); cells expressing insulin-like growth factor 2 (IGF2); cells expressing leukemia inhibitory factor (LIF); cells expressing bone morphogenetic protein 4 (BMP4); cells expressing BMP endothelial cell precursor-derived regulatory factor (BMPER); cells expressing left-right determining factor 1 (LEFTY1); or combinations thereof.

[0014] In one or more embodiments of either method, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0015] In one or more embodiments of either method, the culture vessel or cell culture device is free of a biomimetic coating.

[0016] In another aspect, the disclosure describes a method of treating a subject having or at risk of having a disease involving degeneration of articular cartilage. Generally, the method comprises administering to the subject a composition comprising chondrogenic MTO-derived material in an amount effective to ameliorate at least one symptom or clinical sign of the disease.

[0017] In one or more embodiments, the MTO-derived material comprises chondrocytes, chondrocytes, or both.

[0018] In one or more embodiments, the chondrogenic MTO-derived material is administered in an amount effective to promote regeneration of hyaline cartilage, promote production of type II collagen, reduce the presence of bone spurs, reduce joint pain, reduce joint inflammation, or any combination of two or more of the foregoing.

[0019] In one or more embodiments, the disease is osteoarthritis, cartilage damage, intervertebral disc disease, rheumatoid arthritis, hemochromatosis, psoriatic arthritis, gout, axial spondyloarthritis, or juvenile arthritis, Saldino achondrogenesis, hypochondroplasia, lethal spondyloarthritis, Torrance congenital spondyloepiphyseal dysplasia, Kniest dysplasia, SED with shortened metatarsals, Czech dysplasia, spondylodistal ... Physeoepiphyseal dysplasia (SEMD), Strudwick type, Stickler syndrome type 1, mild SED with early-onset arthropathy, osteochondritis dissecans, relapsing polychondritis, chondrocalcinosis, osteochondroma, enchondroma, periosteal chondroma, multiple chondromatoses, enchondromatosis, chondroblastoma, chondromyxofibroma, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthritis, or temporomandibular joint disorders.

[0020] In another aspect, the disclosure describes a method of treating a subject having or at risk of having a disease involving degeneration of neural cells or neural tissue. In general, the method comprises administering to the subject a composition comprising a neural cell MTO-derived material in an amount effective to ameliorate at least one symptom or clinical sign of the disease.

[0021] In one or more embodiments, the neuronal MTO derived material is administered in an amount effective to promote formation of neural rosettes in a subject, promote formation of neural progenitor cells in a subject, increase dopaminergic neurons in a subject, increase mature astrocytes in a subject, increase oligodendrocytes in a subject, increase markers of cerebral cortex formation in a subject, promote engraftment of transplanted cells in brain tissue of a subject, increase migration of transplanted cells in brain tissue of a subject, reduce the severity and / or extent of a symptom or clinical sign of a neurological disorder, or any two or more of the foregoing.

[0022] In one or more embodiments, the disease is Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, spinal muscular atrophy, progressive supranuclear palsy, multiple system atrophy, or stroke.

[0023] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly illustrates exemplary embodiments. In several places throughout this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each example, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief description of the drawings]

[0024] [Figure 1]Histological characterization of hyaline cartilage formation in multi-tissue organoids (MTO). Histology of 1024 induced pluripotent stem cell (iPSC) line-derived multi-tissue organoids (MTO) at 8 and 30 weeks, and 9-1 iPSC cell line-derived MTO at 11 weeks (Lindborg et al., Stem Cells Transl Med 5(7):970-979 (2016)). (A) 1024 iPSC cell line-derived multi-tissue organoids (MTO) at 8 weeks show developing cartilage nodules with diffuse Alcian blue staining of early cartilage matrix (blue), diffuse labeling of aggrecan (ACAN, brown) in cells and matrix, and type II collagen labeling (COL2A1, brown) in the cartilage matrix in the central region of mature cartilage. Size bar = 200 μm. (B) MTO from 9-1 iPSC cell line at 11 weeks shows mature cartilage with increasing Alcian blue positive cartilage matrix separating chondrocytes, moderate diffuse staining for collagen type II and diffuse aggrecan labeling. Size bar = 50 μm. (C) MTO from 1024 iPSC cell line after 30 weeks in culture shows further maturation to hyaline cartilage morphology where chondrocytes are surrounded by abundant matrix with diffuse Alcian blue and collagen type II staining, and pericellular aggrecan staining. Size bar = 50 μm. (D) Low magnification view of 1024 iPSC cell line MTO at 30 weeks shown in (C) showing that some cartilage nodules have reached a large size. Measurement bar for H&E panel = 4363 μm (4.363 mm). Size bar = 1000 μm. (E) Histomorphometry on tissue sections of MTO from 1024 iPSC cell line using aggrecan area percentage in MTO at 8 and 11 weeks (** indicates p=0.002).

[0025] [Figure 2A]Cartilage development in the MTO is associated with prioritized bone morphogenetic protein (BMP) signaling pathways and increased mesodermal gene expression. (A) Principal component analysis (PCA) of the MTO global transcriptome at 8, 11, and 15 weeks. (B) Differential gene expression analysis showing cartilage marker genes that are differentially expressed between 8 and 15 weeks. (C) Ontology enrichment of genes that are differentially decreased between 8 and 15 weeks. [Figure 2B] Cartilage development in the MTO is associated with prioritized bone morphogenetic protein (BMP) signaling pathways and increased mesodermal gene expression. (A) Principal component analysis (PCA) of the MTO global transcriptome at 8, 11, and 15 weeks. (B) Differential gene expression analysis showing cartilage marker genes that are differentially expressed between 8 and 15 weeks. (C) Ontology enrichment of genes that are differentially decreased between 8 and 15 weeks. [Figure 2C] Cartilage development in the MTO is associated with prioritized bone morphogenetic protein (BMP) signaling pathways and increased mesodermal gene expression. (A) Principal component analysis (PCA) of the MTO global transcriptome at 8, 11, and 15 weeks. (B) Differential gene expression analysis showing cartilage marker genes that are differentially expressed between 8 and 15 weeks. (C) Ontology enrichment of genes that are differentially decreased between 8 and 15 weeks.

[0026] [Figure 3A]Cartilage development in MTO is associated with prioritized BMP signaling pathways and increased mesodermal gene expression. (A) Ontology enrichment of genes significantly increased between 8 and 15 weeks. (B) Table showing comparison of global expression of grouped genes between 8 and 15 weeks and statistical results. Number of expressed genes / number of genes associated with GO terms (searched in biomaRt v2.46.3); one-tailed Wilcoxon test comparing normalized and log-transformed transcript counts between 8 and 15 weeks. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001. [Figure 3B] Cartilage development in MTO is associated with prioritized BMP signaling pathways and increased mesodermal gene expression. (A) Ontology enrichment of genes significantly increased between 8 and 15 weeks. (B) Table showing comparison of global expression of grouped genes between 8 and 15 weeks and statistical results. Number of expressed genes / number of genes associated with GO terms (searched in biomaRt v2.46.3); one-tailed Wilcoxon test comparing normalized and log-transformed transcript counts between 8 and 15 weeks. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.

[0027] [Figure 4] Cartilage development in the MTO is associated with prioritized BMP signaling pathways and increased mesodermal gene expression. Expression of genes encoding molecules commonly used to induce chondrogenesis in vitro in 8-, 11-, and 15-week MTOs.

[0028] [Diagram 5] Schematic summary of findings and associated pathways (not greyed out).

[0029] [Figure 6]Cartilage development in MTO is associated with distinct Wnt and TGF-β / BMP signaling. 125 genes were selected (FDR<0.05) and plotted for scaled time course expression at 8, 11, and 15 weeks, comparing gene expression at 8 and 15 weeks. ****p<0.0001. (Left); Genes arranged by decreased (top left) and increased (bottom left) expression. Functional association network of protein production expressed by genes with decreased expression (top right). Functional association network of protein production expressed by genes with increased expression (bottom right).

[0030] [Figure 7A] The transcriptional signature in MTO is comparable to that in human lower limb chondrocytes. (A) Principal component analysis (PCA) of 325 chondrocyte-specific genes in MTO and human lower limb chondrocytes. (B) Pearson correlation plot of 325 chondrocyte-specific genes in MTO and human lower limb chondrocytes. [Figure 7B] The transcriptional signature in MTO is comparable to that in human lower limb chondrocytes. (A) Principal component analysis (PCA) of 325 chondrocyte-specific genes in MTO and human lower limb chondrocytes. (B) Pearson correlation plot of 325 chondrocyte-specific genes in MTO and human lower limb chondrocytes.

[0031] [Figure 8-1] The transcriptional signature in MTO is comparable to that of human lower limb chondrocytes. Representative comparison of marker transcripts (COL2A1, COL9A1, COL6A2, COL11A1, COL10A1, MMP13, ACAN, CD44, PGR4) in MTO and human lower limb chondrocytes. MTO, multi-tissue organoid; GPC, growth plate chondrocytes. [Figure 8-2] The transcriptional signature in MTO is comparable to that of human lower limb chondrocytes. Representative comparison of marker transcripts (COL2A1, COL9A1, COL6A2, COL11A1, COL10A1, MMP13, ACAN, CD44, PGR4) in MTO and human lower limb chondrocytes. MTO, multi-tissue organoid; GPC, growth plate chondrocytes. [Figure 8-3] The transcriptional signature in MTO is comparable to that of human lower limb chondrocytes. Representative comparison of marker transcripts (COL2A1, COL9A1, COL6A2, COL11A1, COL10A1, MMP13, ACAN, CD44, PGR4) in MTO and human lower limb chondrocytes. MTO, multi-tissue organoid; GPC, growth plate chondrocytes.

[0032] [Figure 9] Rat model of osteoarthritis. Hematoxylin and eosin staining of knee joints treated with organoid-derived chondrocytes and untreated knee joints. The top image (untreated control) and bottom image (treated with chondrogenic organoids prepared without hydrogel) were scored using a scale of 0 to 12 according to the Osteoarthritis Research Society International (OARSI) method, with 12 indicating severe osteoarthritis.

[0033] [Figure 10] Chondrocyte engraftment in a rat model of osteoarthritis. Antibody specific for human Ku80 (arrow) confirms engraftment of human organoid-derived chondrocytes in a treated rat model of osteoarthritis. No Ku80 staining was observed in control joints.

[0034] [Figure 11]Goat model of cartilage damage repair. Cartilage damage was surgically induced by an 8 mm diameter full-thickness injury in the non-weight-bearing part of the cartilage of the stifle (knee) joint of two goats. At the time of surgery, the lesions were treated as follows: In goat #1 (male), the treated side received single-cell chondrocyte product derived from MTO-derived chondrocyte spheres embedded in fibrin glue (TISSEEL, Baxter International Inc., Deerfield, IL), and the control side received fibrin glue alone. In goat #2 (female), the treated side received MTO-derived chondrocyte spheres embedded in fibrin glue, and the control side received fibrin glue alone. Twelve weeks after surgery, the goats were sacrificed and the knees were examined for gross pathology, then fixed in neutral buffered formalin (NBF) and subjected to histopathological examination. Gross pathology (left) as well as hematoxylin and eosin staining (right) show increased proliferation in the treated lesions compared to the control joints. These results show significant neocartilage formation in the treated lesions and minimal or no regeneration in the control lesions.

[0035] [Figure 12]Cartilage damage was surgically induced by an 8 mm diameter full-thickness injury in the non-weight-bearing part of the cartilage of the stifle joint in two goats. At the time of surgery, the lesions were treated as follows: In goat #1 (male), the treated side received a single-cell chondrocyte product derived from MTO-derived chondrocyte spheres embedded in fibrin glue (TISSEEL, Baxter International Inc., Deerfield, IL), and the control side received fibrin glue alone. In goat #2 (female), the treated side received MTO-derived chondrocyte spheres embedded in fibrin glue, and the control side received fibrin glue alone. Twelve weeks after surgery, the goats were sacrificed and the knees were examined grossly pathologically, then fixed in neutral buffered formalin (NBF) and submitted for histopathological examination. Alcian blue stained tissue (upper panel - blue) shows the proteoglycan / hyaluronic acid components typical of cartilage. Collagen type II immunohistochemistry (bottom panel - brown) also shows significant amounts of the specific cartilage marker protein, collagen type II, in the regenerated articular cartilage of treated joints in both subjects. These results indicate significant neocartilage formation in the treated lesions and minimal or no regeneration in the control lesions.

[0036] [Figure 13] Histology of the MTO at 2 weeks showing neural rosettes (arrows) in the top left panel, neural tube-like structures (arrowheads) in the top middle panel, and organized neuroepithelial regions (asterisks) in the top right panel. The bottom panel shows extensive labeling of the MTO with the neural marker β-3 tubulin.

[0037] [Figure 14] Immunofluorescence staining of MTO at 2 weeks. Neural rosettes (arrows) show cells with double immunofluorescence labeling for SOX1 / nestin and SOX2 / nestin, consistent with neural progenitor / stem cells present in the rosettes.

[0038] [Figure 15]Immunohistochemistry of MTO at 2 weeks. Immunohistochemistry staining shows expression of several neuronal markers (brown staining areas) including beta-3 tubulin (β-3 tubulin), Sox2, nestin, Nurr1, and Pax6. Tyrosine hydroxylase, a marker of dopaminergic neurons, is also shown (arrow). A hallmark pathology of Parkinson's disease is the loss of dopaminergic neurons in the substantia nigra.

[0039] [Figure 16] Immunohistochemistry of 2-week MTO. Immunohistochemistry staining for Olig2. Olig2 is a marker for oligodendrocytes and motor neuron precursors (brown staining) and is present in the neuroepithelial region of 2-week MTO.

[0040] [Figure 17] Immunofluorescence staining of MTO at 4 weeks. Immunofluorescence staining showing three markers of cortical formation including Tbr1, doublecortin, and reelin (green). Cell nuclei were stained blue with To-Pro-3.

[0041] [Figure 18] Immunohistochemistry of MTO tissue sections from week 6 onwards. MTO tissue sections stained by immunohistochemistry showed further maturation of neurons and glia, as evidenced by expression of GFAP (red) for astrocytes, MAP2 (brown) for neurons and MBP (brown) for oligodendrocytes.

[0042] [Figure 19] Engraftment sites of 6-week-old human MTO-derived cells are shown by STEM121 immunohistochemistry staining in two rats 8 weeks after surgery. Brown-stained areas show positive staining for STEM121, indicating the presence of human cells. In higher magnification images (right panel), thin processes of STEM121-positive cells can be seen extending into adjacent regions of the rat brain, consistent with neural engraftment.

[0043] [Figure 20]Engraftment sites of 6-week-old human MTO-derived cells are shown by STEM121 immunohistochemical staining (brown stained areas indicate positive staining for STEM121) in two rats at 8 weeks post-surgery. Staining was also present on the contralateral side of the brain where the cells were injected, indicating extensive migration of transplanted cells within the brain.

[0044] [Figure 21] Rat brain (striatum) 8 weeks after surgery transplanted with 6-week-old human MTO-derived cell products. (A) Co-localization of myelin basic protein (MBP) (green) and STEM121 (red) indicates engrafted human oligodendrocytes. (B) Co-localization of microtubule-associated protein 2 (MAP2) (green) and STEM121 (red) indicates engraftment of human neural cells.

[0045] [Figure 22] Therapeutic effect of 6-week-old human MTO-derived neuronal cells measured by amphetamine-induced rotations (rotations per minute). Nude rats with induced parkinsonism were treated with 300,000 6-week-old MTO-derived cells injected into the striatum. Baseline assessments were performed before MTO cell treatment and at 2, 4, 6, and 8 weeks post-surgery. High rotations / min (>10) indicate parkinsonism. B2 is a normal control (non-parkinsonian) rat. The therapeutic effect (reduced rotations) can be seen in rats B4, B7, B8, and B11 over the course of the study (rat 11 died after 2 weeks from an infection unrelated to the cell product).

[0046] [Figure 23] MTOs derived from iPSC line 1024, generated without the use of a 3D matrix, were harvested at 16 weeks and immunohistochemically stained for GFAP as a marker for astrocytes. Reddish-brown stained cells indicate positive staining. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] This disclosure describes a novel method for growing iPSC-derived multi-tissue organoids (MTO), which allows MTO to spontaneously produce therapeutic substances.Therapeutic substances include, but are not limited to, chondrocytes that produce hyaline cartilage, chondrospheres, and neural tissue.

[0048] Organoids are three-dimensional (3D) cultures that realize the self-organizing potential of stem cells. Transcriptome analysis has shown that organoids can recapitulate various early developmental processes of human organs, including the brain, retina, kidney, intestinal epithelium, and villous blasts. As the highest level of cartilage tissue formation occurs during fetal development and early postnatal life, 3D organoid systems are uniquely capable of in vitro cartilage development and the organization of the associated extracellular matrix. For clinical application of organoid-derived hyaline cartilage and chondrocytes in humans, xenobiotic-free, serum-free culture protocols are required. In the future, elucidation of the key mesoderm formation pathways associated with cartilage production in organoids is necessary to facilitate cartilage production by organoid engineering.

[0049] Exemplary Therapeutic Areas of Use Osteoarthritis One area where MTO-derived materials are expected to be of clinical utility is in the treatment of osteoarthritis. The exact pathogenesis of osteoarthritis remains unclear. Osteoarthritis was initially thought to be an inevitable, passive, age-related disease caused by biomechanical factors. It is now widely accepted as a dynamic, multifaceted process involving inflammatory, mechanical, and metabolic factors that create an imbalance between destruction and repair of joint tissue, resulting in the inability of the articular surface to absorb and distribute mechanical loads through the synovial joint. Osteoarthritis is clinically manifested by synovial swelling and inflammation, thin, rough articular cartilage, and / or reactive bone hyperplasia at the joint margins and beneath the cartilage. On radiographs, joint space narrowing (JSN), osteophytosis, subchondral sclerosis, cyst formation, and / or abnormalities of the skeletal contour can also be seen. Osteoarthritis is not limited to cartilage or subchondral bone. Rather, osteoarthritis results from interactions between tissues of the osteochondral complex, including fat and synovial tissue, and the ligaments, tendons, and muscles that surround the joints. However, no disease-modifying drugs for osteoarthritis have been shown to control or reverse osteoarthritis.

[0050] iPSC-derived MTO offers the potential for a disease-modifying therapy for osteoarthritis to repair and restore cartilage, reduce inflammation, and / or significantly delay or reduce the need for joint replacement.

[0051] cartilage damage repair Another area where MTO-derived materials are expected to be of clinical utility is in the repair of cartilage damage. Cartilage tears and osteochondral defects involving articular cartilage are common injuries that can lead to joint replacement. Damaged articular cartilage has a limited ability to self-regenerate, and there are limited treatment options for symptomatic cartilage lesions. Surgical repair methods (e.g., osteochondral auto / allografting and bone marrow stimulation with microfracture) all have limitations, including a lack of suitable grafts and / or failure to obtain repair tissue composed of hyaline cartilage rather than fibrocartilage. To circumvent the limitations of surgical repair methods, cell-based methods have been explored to treat articular cartilage damage. First and second generation autologous chondrocyte transplantation procedures have resulted in good patient outcomes, but the morbidity and costs associated with surgical harvesting of autologous chondrocytes for in vitro expansion remain limitations to the widespread clinical use of chondrocyte transplantation methods.

[0052] To circumvent the limitations associated with surgical harvesting of autologous cultured chondrocytes, in vitro chondrogenesis and hyaline cartilage production from human stem cells has been explored.

[0053] Neurodegenerative disorders Yet another area of ​​clinical utility for MTO-derived substances is the treatment of chronic and / or progressive neurological disorders, such as Parkinson's disease. Affected individuals typically have hand tremors, rigidity, slowness of movement, and postural instability. As the disease progresses, clinical signs worsen and include gait stomp (short walking and stooped posture), speech difficulties (slurred speech), difficulty swallowing, mask-like facies, and cognitive decline. Patients eventually become bedridden and unable to move or care for themselves, resulting in expensive hospital and nursing home costs. Currently available treatments for Parkinson's disease (pharmacotherapy and deep brain stimulation) attempt to compensate for the loss of dopamine (DA) by providing chemical precursors or stimulating brain areas that respond directly to the midbrain cells that produce dopamine. Unfortunately, these treatments are primarily symptomatic, eventually lose efficacy, can have significant side effects, and do not prevent disease progression.

[0054] Preparation and characterization of multi-tissue organoids Induced pluripotent stem cells (iPSCs) have the potential to differentiate into chondrocytes. However, there are still several challenges for the clinical application of iPSC-derived chondrocytes. A common approach to generate iPSC-derived chondrocytes has been to use 2D-3D sequential culture, in which iPSC-derived mesodermal cells are cultured in monolayer and then transferred to 3D cell culture. Introducing 3D culture to this process improves the quality of the cartilage generated. Nevertheless, these existing stepwise protocols are laborious, involve the use of fetal bovine serum (FBS), and involve the manipulation of inductive and inhibitory signals for mesoderm specification during embryonic development. Furthermore, the long-term maintenance and outcomes of chondrocytes in suspension and pellet cell cultures remain to be elucidated.

[0055] Organoid-derived chondrogenic material To gain a deeper understanding of the molecular pathways of MTO during in vitro cartilage production, we performed RNA-seq at 8, 11, and / or 15 weeks after MTO induction. We compared our RNA-seq data with existing RNA-seq data from human chondrocytes at different life stages, and the results showed that relevant gene expression in 15-week MTO was strongly correlated with human fetal lower limb tissues.

[0056] Chemically defined xeno-free culture In one aspect, the present disclosure describes the spontaneous generation of human hyaline cartilage from hiPSC-derived MTO cultured in xeno-free and feeder-free protocols. As used herein, the term "xeno-free" refers to a culture system designed to contain only components derived from the same species as the iPSC. Thus, while human hyaline cartilage is described herein in the context of an exemplary embodiment in which human iPSC (hiPSC)-derived MTO is produced in a xeno-free system containing only human components, xeno-free systems based on the use of iPSCs derived from other species are limited to having only components derived from the species from which the iPSCs were derived. As used herein, the term "feeder-free" refers to a culture system that does not use feeder cells, typically fibroblasts.

[0057] Overcoming the limitations of traditional methods As described above, a method for generating cerebral organoids (COs) from human pluripotent stem cells using a chemically defined hydrogel material (CELL-MATE3D, BRTI Life Sciences, Two Harbors, MN) and culture medium (E8), as well as characterization of the organoids, have been previously reported (Lindborg et al., Stem Cells Transl Med 5(7):970-979 (2016); US Patent Publication No. 2020 / 0248139(A1)). The culture period of these organoids was limited due to central necrosis of the organoids, possibly due to hypoxia, when the organoids reached a diameter of 2-3 mm.

[0058] To address this limitation, the present disclosure describes a method that uses a bioreactor system (G-REX 100, Wilson Wolf, New Brighton, MN) that has a gas-permeable bottom, thereby allowing oxygen diffusion from the bottom as well as the top of the culture medium interface. Using this system with continuous use of E8 medium alone, organoids can be routinely cultured for several months (e.g., up to 30 weeks). The method described herein further modifies a previously reported method by eliminating the use of the chitosan component of CELL-MATE3D (BRTI Life Sciences, Two Harbors, MN). Although the cerebral phenotype of the organoids remained prominent, cartilage-like tissue developed. The cartilage formed in the center of the MTO was easily recognizable histologically from its distinct morphology and characteristic histochemical and immunohistochemical features. The main types of cartilage (articular cartilage, hypertrophic cartilage, elastic cartilage, and fibrocartilage) can be distinguished by the structure and composition of their extracellular matrix (ECM). For example, articular cartilage has a hyaline-like morphology rather than a fibrous morphology (fibrocartilage), contains primarily type II collagen, little or no type I collagen (fibrocartilage) or type X collagen (hypertrophic cartilage), and contains no elastic fibers. By 8 weeks, hyaline cartilage was clearly identifiable in the MTO, as shown by the development of a characteristic abundant, homogenous, pale basophilic extracellular matrix (ECM) in H&E stained sections (Figure 1A), and prominent Alcian blue staining of proteoglycan / hyaluronic acid components typical of cartilage ECM (Figure 1B). Immunohistochemistry of MTO cartilage showed that the amount of type II collagen increased over time as the cartilage matured, and showed widespread expression of aggrecan at all time points (Figure 1C,D). Type VI collagen was also widely expressed in MTO cartilage, but type I collagen showed expression at a few sites in the periphery of MTO cartilage, and type X collagen showed no immunoreactivity above background overall. The hyaline cartilage phenotype was stably maintained from week 8 to week 30, and it grew significantly in size (Figures 1A to 1D).To further evaluate this, histomorphometry was performed on MTO tissue sections using aggrecan area percentage as an overall index of developing and mature cartilage composition in the MTO. At 8 weeks (n=7 biological replicates), aggrecan area percentage was 17.7% (±7.2) and at 11 weeks (n=5) was 57.8 (±15.1), with an unpaired t-test indicating a significant difference (p=0.0001) (Figure 1E).

[0059] Global transcriptome revealed a mesoderm formation signature in the MTO. To understand the transcriptomic changes underlying cartilage phenotypic development in the MTO, we performed bulk RNA-seq of 1024-derived MTOs at weeks 8, 11, and 15, covering the period during which histological analysis showed the emergence, expansion, and maturation of MTO cartilage. Principal component analysis (PCA) of MTO global RNA expression data showed clear clustering corresponding to harvest time (Figure 2A). Differential expression analysis and gene ontology (GO) enrichment of differentially expressed genes were performed for all three comparisons. COL2A1 content was increased in MTO cartilage (Figure 1C), whereas COL2A1 expression was decreased in bulk RNA-seq. This is likely due to the multi-tissue nature of the MTO. However, other cartilage markers such as ACAN, CD44, COMP, PRG4, and SNAI1 showed significantly increased expression over the time course of the experiment (Figure 2B). Transcript levels of collagen I / X were increased, whereas expression of IHH, another hypertrophy marker, was decreased (Figure 2B). In addition, type I collagen is the most abundant collagen and its expression is not restricted to cartilage. Therefore, the increase in the transcript levels of type I collagen in MTO does not necessarily reflect an increase in type I collagen in the composition of MTO cartilage. Interestingly, we observed a consistent downregulation of neural processes such as synaptogenesis, axon formation, and an upregulation of mesodermal processes such as extracellular matrix formation, connective tissue development, and cartilage development (Figure 2C, Figure 3A).

[0060] The interplay between bone morphogenetic proteins (BMPs) and fibroblast growth factor (FGF) signaling plays a highly conserved role in processes including, but not limited to, neural induction and mesodermal patterning, chondrocyte differentiation and proliferation, and endochondral ossification during embryonic development. The gradual increase in cartilage production observed from 8 to 15 weeks suggests that the overall neural to mesodermal transition observed in human MTOs may be associated with a marked change in the dynamics between the BMP and FGF pathways. In detail, the BMP pathway may have been favored with increased expression of components in the BMP signaling pathway, reduced or unchanged levels of BMP antagonists, and / or reduced or constant levels of FGF signaling. Because pathways in the mesoderm developmental process are intertwined and expressed in various tissues, identifying and examining only a few genes expressed in one pathway may lead to bias in bulk sequencing of MTOs. Therefore, we comprehensively examined the expression of genes based on previous publications and GO terms, and compared the overall expression of grouped genes at 8 and 15 weeks. The overall expression of BMP (p=0.0041) and its intracellular signaling molecule SMAD (p=0.00098) was significantly increased (Fig. 3B). The overall expression of BMP antagonists was unchanged (p=0.6) (Fig. 3B). Regarding the FGF signaling pathway, the expression of neural FGF was examined and, although it was not significantly changed (p=0.29), it was expressed at a lower level compared to the other transcripts mentioned above (Fig. 3B). Furthermore, the dynamic expression of other components of the FGFR pathway was examined and revealed that the expression of genes involved in the negative regulation of the FGFR signaling pathway (GO:0040037) was significantly increased (p=0.02), while the expression of genes involved in the positive regulation of the FGFR signaling pathway (GO:0045743) was decreased, although not statistically significant (p=0.097) (Fig. 3B). This suggests that neural FGF and its downstream pathways were suppressed, which is consistent with the observed histological decrease in neural components.Finally, we examined the expression dynamics of genes involved in mesoderm formation (GO:0001707), revealing a significant increase (p<0.0001) in the expression of genes involved in this biological process (Figure 3B).

[0061] Since the chondrogenic phenotype was spontaneously favored by MTO without the addition of any chemicals other than E8 medium, MTO may inherently increase the expression of differentiation factors used to induce chondrogenesis. Expression of BMP2, BMP6, FGF2, and TGFB1 was significantly increased spontaneously in MTO from week 8 to week 15 (Figure 4). In addition, there was expression of other genes whose products are used for chondrogenesis in other protocols investigated, including TGFB3, INHBA, BMPER, BMP4, LIF, IGF2, LEFTY1, SFRP1, and CER1. All expression patterns except CER1 were increased (Figure 4). This indicates that the molecular mechanisms underlying spontaneous chondrogenesis in MTO may be similar to those observed in hiPSC-induced chondrogenesis.

[0062] Overall, the global transcriptome of hiPSC-derived MTOs showed that increased gene expression in BMP signaling and mesoderm development and decreased gene expression in neural FGF signaling were associated with increased cartilage production in MTOs from weeks 8 to 15 (Figure 5).

[0063] Distinct signaling pathways associated with increased articular cartilage development in MTO The establishment of both chondrogenesis and osteogenesis is a consequence of cartilage development. Chondrogenesis plays a key role in the fetal development of the mammalian skeletal system. Chondrocyte hypertrophy and cartilage matrix degradation precede endochondral ossification, which results in the formation and growth of long bones. Because chondrogenesis and osteogenesis are integral, gene expression for chondrogenesis and ossification largely overlaps. As mentioned earlier, we observed an increase in several cartilage marker genes, as well as reciprocal expression of IHH and COL10A1 in MTO (Figure 1). However, no degradation of the cartilage matrix, mineralization, or ossification was observed even at 30 weeks. Therefore, we investigated the temporal expression patterns of genes involved in cartilage development to determine whether we could identify signaling pathways that may contribute to the long-term maintenance of chondrocytes. To accomplish this, we first examined gene expression in the GO term “cartilage development” (GO:0051216), which includes both positive and negative regulators in cartilage development. When the expression patterns of 125 genes (FDR<0.05) out of 194 genes searched by biomaRt were visualized, they were clearly separated into 42 genes whose expression was decreased and 83 genes whose expression was increased (Figure 6). In addition, despite the decrease in the expression of 42 genes, the overall gene expression level significantly increased with cartilage development (p<0.0001), consistent with the expansion of cartilage in the MTO (Figure 3, left panel).

[0064] STRING was then used to construct two functional association networks consisting of proteins expressed by the described genes to identify distinct interactions of gene products of selected upregulated and downregulated genes. Transcription factors and growth factors were the most highly associated molecules in both networks. Wnt signaling molecules constituted the most prominent local network cluster in the network clustering for downregulated gene products (Figure 6, top right panel; Table 1).

[0065] [Table 1]

[0066] The Wnt signaling cascade has important roles in the development and homeostasis of cartilage development and ossification. In general, the canonical Wnt cascade (including ROR2 and SFRP2, which are featured in the network) suppresses the early stages of cartilage development. Overexpression of WNT7A inhibited early cartilage development in a chick limb model. In endochondral ossification, the Wnt signaling pathway promotes chondrocyte hypertrophy. SFRP2, WNT01B, and WNT7B are known positive regulators of ossification. In addition, several non-Wnt positive regulators of ossification were also downregulated. Among the upregulated members, a clustering was observed around the TGF-β / BMP signaling pathway (Figure 6, lower right panel). We also observed a unique signature of increased downregulation of the canonical Wnt signaling pathway, consistent with an association network formed by the protein products of genes with reduced expression. In contrast to the previous network, some negative regulators of ossification had little overlap with the TGF-β / BMP signaling pathway (Figure 6, lower right panel). In summary, the cartilage in MTOs was stably developed from week 8 to week 15 due to the continuous increase in the TGF-β / BMP pathway, which promotes chondrogenesis, and the downregulation of the Wnt signaling cascade, which suppresses chondrocyte hypertrophy and ossification in mature chondrocytes.

[0067] In addition, to reduce the bias of gene selection, the expression of genes in the biological processes of interest was also compared as above using the GO term gene list retrieved from GO by biomaRT (Table 2 ).

[0068] [Table 2] 1 Number of expressed genes / number of genes related to GO terms (searched using biomaRt v2.46.3) 2 One-sided Wilcoxon test comparing normalized and log-transformed transcript counts between weeks 8 and 15 3* is p value < 0.05; **p-value <0.01.

[0069] Again, genes in GO terms related to promoting cartilage maturation, including positive regulation of cartilage development (GO:0061036), chondroblast differentiation (GO:0060591), and cartilage morphogenesis (GO:0060536), showed significantly increased expression (Table 2), whereas the growth plate cartilage development gene set (GO:0003417) did not increase significantly (p=0.058). Furthermore, expression of genes related to negative regulation of chondrocyte maturation and promotion of endochondral ossification did not show significant changes (Table 2). Since expression of growth plate cartilage development (GO:0003417) marker genes was only moderately increased (p=0.058), it is possible that the growth of hyaline cartilage produced by MTO is more closely associated with increased expression of articular cartilage gene markers, but not others. Indeed, a significant increase in the expression of articular cartilage marker genes (GO:0061975; p = 0.031) was observed, but not bronchial or tracheal cartilage development (GO:0060532; GO:0060534).

[0070] In summary, the protein products of genes whose expression was decreased in MTO from weeks 8 to 15 were clustered around components of the Wnt signaling pathway that promotes ossification, whereas the protein products of genes whose expression was increased formed a prominent network around the TGF-β / BMP signaling pathway. Furthermore, the observed molecular signaling clusters were associated with unique increases in gene expression for articular cartilage development.

[0071] Transcriptome comparison between MTO chondrocytes and lower limb chondrocytes across human life stages Because MTO RNA-seq revealed unique increases during articular cartilage development, there may be strong correlations between MTO chondrocytes and human growth plate chondrocytes and / or articular chondrocytes in the expression of genes specific to human growth plate chondrocytes.We reprocessed existing RNA-seq data collected from human embryonic limb buds (week 6; Li et al., BMC Genomics 18(1):983(2017)), growth plate chondrocytes (weeks 14, 15, 16, and 18; Li et al., BMC Genomics 18(1):983(2017)), knee chondrocytes (week 17, adolescent (Hicks et al., Nat Cell Biol 20:46-57(2018)), and adult (Ferguson et al., Nat Commun 9(1):3634(2018)), and costal chondrocytes (adults aged approximately 70 years). We examined 325 genes known to be specifically expressed in chondrocytes and performed a principal component analysis (PCA) on these genes. Gene expression was clustered by life stage, not by specific study, so that the influence of different studies is minimal (Figure 7A). In detail, fetal tissues (growth plate and knee chondrocytes) clustered together, while adolescent, adult, and 70-year-old cartilage tissues (knee chondrocytes and costal chondrocytes), collectively referred to as post-in utero tissues, were located close to each other (Figure 7A). The 15-week MTO was most similar to 6-week human limb bud cartilage tissue (Figure 7A). Pearson correlation coefficients were then used to examine the correlation between all samples and genes included in the PCA analysis. All MTOs showed a correlation of over 60% with human chondrocytes and cartilage tissues from all life stages, while the 15-week MTO showed an even stronger correlation (over 76%) with 6-week human limb bud, 14-week fetal growth plate chondrocytes, and 15-week fetal growth plate chondrocytes (Figure 7B). Furthermore, MTO showed a significantly higher (p<0.0001; 95% CI[0.053,+∞]) mean correlation with fetal chondrocytes than with post-in utero knee chondrocytes for the previously described genes of interest. The mean correlation between MTO and fetal tissues was 71%, whereas the mean correlation with post-in utero tissues was 65%.

[0072] The expression of known collagen genes (COL2A1, COL9A1, COL6A2, COL11A1, COL10A1), hypertrophic markers (COL10A1, MMP13), and several components reflecting chondrocyte secretory function (ACAN, CD44, PGR4) were further examined in MTO compared to human hindlimb chondrocytes and cartilage (Figure 8). Transcripts of components constituting the ECM were overall more abundant in in utero hindlimb chondrocytes than in post-in utero hindlimb chondrocytes, whereas transcripts of secretory molecules tended to be more diverse. Expression of genes encoding collagen types 2 / 9 (COL2A1, COL9A1) was low in 15-week MTO, whereas expression from 8-week MTO and 11-week MTO fell within the range defined for human tissues examined. There were no significant differences between MTO and human lower limb cartilage tissue in the expression of other major collagen components (COL6A2 and COL11A1) that compose the nonhypertrophic region of cartilage, or hypertrophic markers (COL10A1, MMP13). Among MTO harvested at different time points, 15-week MTO had slightly lower collagen content and higher hypertrophic gene expression, although within the range of expression in human tissue. Regarding the transcripts of secreted molecules, 15-week MTO showed similar expression to human tissue (PGR4, CD44), except for lower expression of ACAN. The normal expression of PRG4, a gene that encodes a large proteoglycan synthesized by chondrocytes located on the surface of articular cartilage and in a part of the synovial lining cell layer, further supports the spontaneous joint developmental trajectory taken by MTO chondrocytes. In summary, the transcript levels of genes specifically expressed in human growth plate chondrocytes were strongly correlated between 15-week MTO and human lower limb cartilage tissue.

[0073] In vitro chondrogenesis and hyaline cartilage production are necessary to provide chondrocytes for cartilage regeneration therapy and ex vivo osteoarthritis modeling. Despite recent advances in hiPSC-based in vitro chondrogenesis, developing a simple and scalable protocol to generate chondrocytes for therapeutic purposes and understanding the dynamic cell behavior in long-term culture remain to be achieved.

[0074] iPSC-derived MTO for osteoarthritis and degenerative cartilage diseases This disclosure describes the spontaneous development and robust growth of hyaline cartilage in hiPSC-derived MTOs grown in xeno-free and feeder-free culture. The culture does not require the addition of growth or differentiation factors other than those present in the E8 culture medium, making the protocol clinically Good Manufacturing Practice (cGMP) compliant. Furthermore, the process is technically relatively simple, making it amenable to robotic cell culture and large-scale manufacturing. By characterizing and analyzing the transcriptomic changes during the phenotypic transition of MTOs, we provide a mechanistic basis for the development of future organoid and related tissue engineering aimed at rapid cartilage production.

[0075] Although described herein in the context of an exemplary embodiment in which the iPSCs used to generate the MTO are human iPSCs (hiPSCs), the methods described herein can include the use of iPSCs derived from other species, for example where it is desired that the MTO produce cartilage material derived from a non-human species.

[0076] Generally, the methods described herein involve culturing iPSCs in a culture medium containing the hydration fluid from the hydrogel-based 3D cell culture matrix, rather than using the 3D hydrogel matrix itself. Culturing iPSCs in a medium containing the hydration fluid is sufficient to induce the iPSCs to form organoids. Generally, the hydration fluid of the hydrogel-based 3D cell culture matrix is ​​a hyaluronic acid solution, containing hyaluronic acid in a concentration range of 0.1% to 1.5%. However, in one or more embodiments, the iPSCs can be cultured in a physiological solution (e.g., normal saline, phosphate buffered saline, Hank's balanced salt solution, DMEM, E8) with or without hyaluronic acid.

[0077] Without wishing to be bound by any particular theory, iPSCs may generate MTO during culture due to factors including, but not limited to, cell signaling that occurs between cultured iPSCs due to the absence of a 3D matrix and / or the cell density of the culture. These factors, rather than any particular culture medium component, may promote the generation of MTO.

[0078] Cartilage in organoids produced by the methods described herein resembles hyaline cartilage, typical of cartilage present during fetal development. Furthermore, hyaline cartilage produced by MTO resembles articular hyaline cartilage in many ways, including collagen type II, collagen type IX, and aggrecan content. For example, the higher the aggrecan content in the ECM, the greater the ability of the cartilage to withstand compression. Thus, cartilage produced by MTO has biomechanical properties similar to articular cartilage, including but not limited to resistance to repeated forces of compression and shear. Thus, cartilage produced with MTO is useful for treating diseases involving degeneration of articular cartilage, including but not limited to cartilage rupture, osteochondral defects, osteoarthritis, etc.

[0079] Figure 9 shows histological data of a rat model treated for osteoarthritis. The top image (untreated control) and bottom image (treated with chondrogenic organoid-derived cells prepared without 3D matrix) were scored using a scale of 0 to 12 according to the method of Osteoarthritis Research Society International (OARSI), with 12 indicating severe osteoarthritis. The scoring method takes into account cartilage thickness, joint cavity thickness, presence or absence of osteophytes, etc. Scoring was performed blinded. The top image, with an OARSI score of 11, shows loss of cartilage and the presence of osteophytes. The bottom image, with an OARSI score of 4, shows mild cartilage erosion, the presence of cartilage, and the absence of osteophytes. Figure 10 shows immunohistochemical staining for Ku80, demonstrating that human organoid-derived chondrocytes engrafted into the treated knee of the rat model.

[0080] cartilage ball Chondrocytes are structures that spontaneously form from chondrocytes dissociated from MTO. Chondrocytes can be obtained by physically dissociating MTO into single cells and then culturing in a bioreactor under conditions where the dissociated chondrocytes spontaneously form spheres of therapeutically active chondrocytes, i.e., chondrocyte spheres. The dissociated cells may or may not be cultured on an ultra-low attachment surface to prevent the chondrocytes from aggregating prior to introduction into the bioreactor.

[0081] Figures 11 and 12 show that human organoid-derived chondrocytes repair damaged cartilage in a goat model of surgically induced articular cartilage injury in the goat femoro-tibial joint. In each goat, a full-thickness defect measuring 8 mm in diameter was created in the articular cartilage of both femoro-tibial joints. In goat #1 (male), the treatment side received single-cell chondrocyte product derived from MTO-derived chondrocytes embedded in fibrin glue (TISSEEL, Baxter International Inc., Deerfield, IL), and the control side received fibrin glue alone. In goat #2 (female), the treatment side received MTO-derived chondrocytes embedded in fibrin glue, and the control side received fibrin glue alone. Twelve weeks after surgery, the treated joints in both goat subjects showed significant regeneration of articular cartilage, as evidenced by the high amount of Alcian blue-stained tissue showing proteoglycan / hyaluronic acid components typical of cartilage (Figure 12). Immunohistochemistry of MTO cartilage also demonstrated significant amounts of the specific cartilage marker protein, type II collagen, in the regenerated articular cartilage of treated joints from both subjects (Figure 12).

[0082] Chondrogenic Products and Methods Thus, the present disclosure describes a scalable and cGMP compatible method for generating authentic hyaline cartilage from chondrocytes and / or chondrocyte spheres derived from organoids generated from human iPSCs. The chondrocytes maintain the chondrocyte phenotype, secrete collagen and anti-inflammatory factors, engraft in damaged joints, and promote repair of cartilage damage in animal models (Figures 9-12). The methods described herein can be scaled up to provide a large volume supply of therapeutic chondrocytes for treating diseases such as, but not limited to, osteoarthritis. Thus, organoid-derived chondrocytes can overcome the shortcomings of current cell-based procedures by providing an off-the-shelf chondrocyte therapy that does not require surgery to harvest patient cells, or expansion of primary cells. Furthermore, the methods described herein produce uniform, high-quality chondrocytes that are not fibroblasts. Finally, the methods described herein use chemically defined materials and are easily adaptable to cGMP manufacturing and robotic scale-up.

[0083] iPSCs are an easily accessible and reproducible source of pluripotent cells that can differentiate into chondrocytes that generate pure cartilage both in vitro and in vivo. Early studies have not shown immune rejection of organoid-derived chondrocytes. Previous approaches to generate iPSC-derived chondrocytes have been limited in their therapeutic application to osteoarthritis due to the complexity and high cost of generating the cells, as well as the large variability associated with the xenogeneic biological agents used in these manufacturing processes. To overcome these shortcomings, the approach described herein generates chondrocytes in bulk from organoids generated from iPSCs without the use of xenobiotics or autologous donor cells.

[0084] Therapeutic potential of iPSC-derived human chondrocytes in musculoskeletal diseases Intra-articular injection of human chondrocytes is a logical therapeutic approach in the treatment of osteoarthritis, but has not yet been put to practical use due to the lack of readily available chondrocytes. Other cell-based approaches using mesenchymal stem / stromal cells may provide short-term effects but have not led to engraftment or long-term repair. Chondrocytes have a strong anti-inflammatory potential that may reduce joint inflammation. Chondrocytes may engraft at the site of cartilage damage and provide long-term repair and clinical improvement, potentially delaying or eliminating the need for knee or hip replacement. Organoid-derived chondrocytes generated from iPSCs are resistant to the inflammatory environment of osteoarthritic joints and are highly similar to juvenile chondrocytes with better proliferation capacity than primary adult chondrocytes. Thus, juvenile chondrocytes may have enhanced survival and engraftment, and long-term efficacy. Importantly, cartilage allografts are known to be well tolerated, and off-the-shelf transplants may be used. The organoid-derived chondrocytes described herein possess all the desirable qualities of juvenile chondrocytes, highlighting their extraordinary potential as orthopedic and surgical therapeutics.

[0085] How to generate organoids to produce cartilage Methods for generating and using organoids to produce cartilage, including culturing iPSCs in a 3D hydrogel matrix, have been described, for example, in International Publication No. WO 2020 / 132055 A1 and Lindborg et al., Stem Cells Transl Med 5(7):970-979 (2016). In those methods, the growth of iPSC-derived multi-tissue organoids (MTOs) is promoted by culturing cells in a 3D hydrogel matrix. These organoids contained primarily brain tissue, but could be engineered to produce greater amounts of hyaline cartilage by controlling inductive and inhibitory signals for mesoderm specification during embryonic development.

[0086] In contrast, the method described herein involves spontaneously growing hyaline cartilage in human iPSC-derived multi-tissue organoids (MTO) without using a 3D hydrogel matrix and without manipulating culture conditions to change the developmental pathway toward a mesoderm-specific direction. Rather, hyaline cartilage is formed spontaneously without a 3D hydrogel cell culture matrix and only using standard cell culture medium (e.g., E8 medium). The method described herein is less laborious and easier to scale up for clinical application because it does not require the use of a 3D hydrogel matrix or serum-based factors to induce mesoderm tissue development in culture conditions. Thus, multi-tissue organoids (MTO) can be grown in a bioreactor and hyaline cartilage contained in the MTO can be isolated. The isolated hyaline cartilage can be disaggregated into individualized cells (e.g., chondrocytes), which can then be used to form chondrocyte aggregates or can be cultured in chondrogenic medium (e.g., on an ultra-low attachment surface).

[0087] Characterization of cartilage-producing organoids Cartilage in MTO was identified by histology and transcriptome analysis. Organoids grown in vitro using the methods described herein contain chondrocytes with gene expression profiles similar to human fetal lower limb tissue. For example, RNA-seq data shows that 8 weeks, 11 weeks, and / or 15 weeks after MTO induction, organoids contain chondrocytes with increased expression of specific genes. Exemplary genes that show increased expression include, but are not limited to, bone morphogenetic proteins (BMPs) and their intracellular signaling factors (SMADs), and differentiation factors that induce chondrogenesis. Thus, exemplary genes whose expression is increased include, but are not limited to, transforming growth factor beta 1 (TGFβ1), fibroblast growth factor 2 (FGF2), bone morphogenetic protein 2 (BMP2), bone morphogenetic protein 6 (BMP6), growth differentiation factor 5 (GDF5), secreted frizzled-related protein 1 (SFRP1), inhibin subunit beta A (INHβA), transforming growth factor beta 3 (TGFβ3), insulin-like growth factor 2 (IGF2), leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), BMP endothelial cell precursor-derived regulatory factor (BMPER), left-right determining factor 1 (LEFTY1).

[0088] In another aspect, the present disclosure describes organoids, organoid cells, organoid tissues, organoid-derived chondrocytes, or organoid-derived chondrocyte aggregates generated using any of the embodiments of the methods described herein.

[0089] In another aspect, the present disclosure describes cartilage produced using multi-tissue organoids grown using any embodiment of the methods described herein. In some cases, the cartilage can be hyaline cartilage or articular cartilage.

[0090] In yet another aspect, the transcriptomic analysis described herein indicates that the developmental dynamics of the chondrogenic pathway allow multi-tissue organoids (MTOs) to serve as a human-specific model for disease modeling and drug testing.

[0091] In summary, one embodiment of the present disclosure describes the long-term culture of iPSC-derived MTOs that result in the spontaneous generation of mesodermally derived articular cartilage tissue that resembles fetal limb bud and growth plate chondrocytes. The process described herein is self-organized using a relatively simple, xenobiotic-free, feeder-free culture protocol, making the production easily adaptable to cGMP manufacturing and suitable for scaled-up commercial production.

[0092] Neuro-MTO products, methods, and expected therapeutic effects In another aspect, the present disclosure describes human midbrain organoid (MBO) derived cell therapy for treating neurological disorders such as Parkinson's disease. Parkinson's disease is a chronic progressive neurological disorder that most significantly affects movement. Currently available Parkinson's disease treatments (pharmacotherapy and deep brain stimulation) attempt to compensate for the loss of dopamine by providing chemical precursors or stimulating brain regions that directly respond to midbrain cells that produce dopamine. Unfortunately, these treatments are primarily symptomatic, and eventually lose efficacy, may cause significant side effects, and / or fail to prevent progression.

[0093] Organoid-Derived Cell-Based Therapies for Neurological Diseases Organoids can also provide a source of cellular therapeutics for neurological diseases. The use of midbrain organoids (MBOs) has several advantages over previously developed neural cell production methods that encourage its use for commercial production. The method is technically simple, does not use animal products, is scalable to produce large quantities of organoids at relatively low cost, does not require time-specific or dose-specific inclusion of growth factors, and is compatible with GMP manufacturing protocols.

[0094] Thus, in one aspect, the present disclosure describes the use of organoids to generate cells that can be used for cell-based regenerative and / or restorative cell therapy to treat neurological disorders such as Parkinson's disease. This therapy may restore dopaminergic neurons and supporting cells lost during the course of this Parkinson's disease, slowing or stopping further disease progression and / or restoring the patient's motor skills. The cell therapy approach described herein may provide subjects with neurological disorders with a higher quality of life while simultaneously reducing the overall cost of treatment.

[0095] Human midbrain organoids (MBOs) involve inducing human iPS cells to form MBOs, which offers several advantages over previously developed neural cell production methods that suggest the use of MBOs for commercial production of neural cells and tissues: the method is technically simple, does not use animal-derived products, is scalable to produce large quantities of organoids at relatively low cost, does not require time-specific and / or dose-specific inclusion of growth factors, and is compatible with GMP manufacturing protocols.

[0096] Although described herein in the context of exemplary embodiments in which the iPSCs used to generate the MTO are human iPSCs (hiPSCs), the methods described herein can include the use of iPSCs derived from other species, for example where it is desired that the MTO produce neural material derived from a non-human species.

[0097] The regenerative and / or restorative cell therapies described herein may restore dopaminergic neurons and supporting cells lost during the course of neurodegenerative disease, slowing or halting further disease progression and potentially restoring motor skills to patients. This cell therapy approach is expected to provide a higher quality of life for patients with neurodegenerative disorders while reducing the overall cost of treatment.

[0098] The brain organoid generation process described herein is robust and consistent. RNA-seq gene expression (2 lines) and immunohistochemical analysis (6 lines) of organoids showed multiple markers for midbrain differentiation. Initial data suggest that MTO-derived cell products may be novel therapeutics for Parkinson's disease. Preclinical data show early formation of neural rosettes, neural tube-like structures, and neural progenitor cells (Sox1 or Sox2 and nestin double-labeled cells) (Figure 13; Figure 14); presence of dopaminergic neurons (tyrosine hydroxylase positive neurons) in MTO derived from two different iPS cell lines (CS1 and R76, Figure 15); presence of oligodendrocyte precursor cells (olig 2 positive cells, Figure 16); presence of cerebral cortex formation markers (T-brain 1, doublecortin and reelin (Figure 17); presence of mature astrocytes (glial fibrillary acidic protein, GFAP), neurons (microtubule-associated protein 2, MAP2), and oligodendrocytes (myelin basic protein, MBP) beyond 6 weeks (Figure 18); engraftment (immunohistochemical staining for STEM121, specific for human cells) of 300,000 MTO-derived cells injected into the striatal region of rat brain at 6 weeks (2 examples shown) at 8 weeks post-transplantation , with extension into the surrounding brain and extensive migration into the contralateral striatum (FIG. 19; FIG. 20), engraftment of human oligodendrocytes (MBP / STEM121 double staining) and human neurons (MAP2 / STEM121 double labeling) was observed (FIG. 21); and therapeutic effects in rats (e.g., reduced rotations) (rat model of Parkinson's disease, FIG. 22), e.g., in rats with chemically induced hemiparkinsonism (similar to FIG. 19, FIG. 20, and FIG. 21).

[0099] Compositions and Treatment Methods Once isolated, the MTO-derived material (whether chondrogenic cells, chondrocytes, or neural cells) can be included in a pharmaceutical composition for administration to a subject. Thus, the present disclosure describes pharmaceutical compositions comprising MTO-derived material. The present disclosure also describes methods of treating a subject having or at risk of having a disease treatable with MTO-derived material.

[0100] The subject can be a human or a non-human animal, such as a livestock animal, a working animal, a laboratory animal, or a companion animal. Exemplary non-human animal subjects include, but are not limited to, animals that are members of the family Hominidae (including, for example, chimpanzees, gorillas, or orangutans), family Bovidae (including, for example, cows), family Capriidae (including, for example, goats), family Ovidae (including, for example, sheep), family Porcineae (including, for example, pigs), family Equidae (including, for example, horses), members of the family Cervidae (including, for example, deer, elk, moose, caribou, reindeer), members of the family Bisonidae (including, for example, bison), family Felidae (including, for example, domesticated cats, tigers, lions, etc.), family Canidae (including, for example, domesticated dogs, wolves, etc.), avian (including, for example, turkeys, chickens, ducks, geese, etc.), rodents (including, for example, mice, rats, etc.), members of the family Leporidae (including, for example, rabbits or hares), members of the family Mustelidae (including, for example, ferrets), or members of the order Chiroptera (including, for example, bats).

[0101] The MTO-derived material described herein may be formulated with a pharma- ceutically acceptable carrier. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and / or agents for pharma- ceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the MTO-derived material, its use in therapeutic compositions is contemplated. In one or more embodiments, supplementary active ingredients may also be incorporated into the composition. As used herein, "pharma- ceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual together with the MTO-derived material without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0102] Thus, the MTO-derived material can be formulated into a pharmaceutical composition. The pharmaceutical composition can be formulated in various forms adapted to the preferred route of administration. Thus, the composition can be administered via known routes, including, for example, orally, parenterally (e.g., intradermally, transdermally, subcutaneously, intramuscularly, intravenously, intraperitoneally, etc.), or topically (e.g., intranasally, intrapulmonary, intramammary, intravaginally, intradermally, transdermally, intrarectally, etc.). The pharmaceutical composition can be administered to mucosal surfaces, for example, by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). The composition can also be administered via sustained or delayed release.

[0103] Thus, pharmaceutical compositions containing MTO-derived substances can be provided in any suitable form, including, but not limited to, any form of solution, suspension, emulsion, spray, aerosol, or mixture. The composition can be delivered in a formulation containing any pharma- ceutically acceptable excipient, carrier, or vehicle. For example, the formulation can be delivered in a conventional topical dosage form, such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, etc. The formulation may further include one or more additives, including, but not limited to, adjuvants, skin penetration enhancers, colorants, fragrances, flavors, moisturizers, thickeners, etc.

[0104] The formulations may be conveniently provided in unit dosage form and may be prepared by methods well known in the art of pharmacy. The method of preparing a composition containing a pharmaceutically acceptable carrier includes the step of combining the MTO-derived material with the carrier, which constitutes one or more accessory ingredients. In general, the formulations can be prepared by uniformly and / or intimately combining the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0105] The amount of MTO-derived material administered may vary depending on a variety of factors, including, but not limited to, the weight, health, and / or age of the subject, and / or the route of administration. Thus, the absolute number of MTO-derived materials contained in a given unit dosage form may vary widely and depends on factors such as the species, age, weight, and health of the subject, and / or the method of administration. Therefore, it is not practical to generally state an amount that constitutes an amount of MTO-derived material effective for all possible uses. However, a person skilled in the art can easily determine an appropriate amount with due consideration of such factors.

[0106] In one or more embodiments, the method may include administering sufficient MTO-derived material to provide a dose of, for example, about 10,000 cells to about 100 trillion cells to the subject, although in one or more embodiments, the method may be practiced by administering MTO-derived material at a dose outside this range. In reference to chondrocytes, the term "cell" number in a dose refers to the number of individual chondrocytes that form the chondrocytes, not the number of chondrocytes.

[0107] In one or more embodiments, the methods may include administering sufficient MTO-derived material to provide a minimum dosage of, for example, at least 10,000 cells, at least 50,000 cells, at least 100,000 cells, at least 200,000 cells, at least 300,000 cells, at least 400,000 cells, at least 500,000 cells, at least 1 million cells, at least 2 million cells, at least 10 million cells, at least 20 million cells, at least 50 million cells, at least 100 million cells, at least 500 million cells, at least 1 billion cells, at least 5 billion cells, at least 10 billion cells, at least 50 billion cells, or at least 100 billion cells.

[0108] In one or more embodiments, the methods may include administering sufficient MTO-derived material to provide a maximum dosage of, for example, 100 trillion cells or less, 50 trillion cells or less, 10 trillion cells or less, 1 trillion cells or less, 500 billion cells or less, 100 billion cells or less, 50 billion cells or less, 10 billion cells or less, 1 billion cells or less, or 500,000 cells or less. Cells are said to be present in an amount "up to" a reference amount if they are present in an amount up to the reference amount, rather than being absent.

[0109] In one or more embodiments, the method may include administering sufficient MTO-derived material to provide a dosage characterized by a range having endpoints defined by any minimum dosage amounts identified above and any maximum dosage amounts identified above that are greater than the selected minimum dosage amount. Thus, in one or more embodiments, the method may include administering sufficient MTO-derived material to provide a dosage of, for example, 100,000 cells to 500,000 cells, 300,000 cells to 1 billion cells, 1 billion cells to 1 trillion cells, 1 million cells to 10 trillion cells, 100 million cells to 500 billion cells, 50 billion cells to 10 trillion cells, etc.

[0110] In certain embodiments, the methods may include administering sufficient MTO-derived material to provide a dosage equal to any minimum dosage or any maximum dosage listed above. Thus, for example, the methods may include administering sufficient MTO-derived material to provide a dosage of 300,000 cells, 500,000 cells, 1 million cells, 1 billion cells, 10 billion cells, 50 billion cells, 100 billion cells, 1 trillion cells, etc.

[0111] A single dose may be administered all at once, continuously for a period of time, or in multiple separate doses. When administered in multiple doses, each dose may be the same or different. For example, a daily dose of 100 billion cells may be administered as a single dose of 100 billion cells, as two or more equal doses (e.g., two doses of 50 billion cells) consecutively over a 24-hour period, or as two or more unequal doses (e.g., one dose of 75 billion cells followed by a second dose of 25 billion cells). When multiple doses are administered to deliver a single dose, the intervals between doses may be the same or different.

[0112] In one or more embodiments, the MTO-derived material may be administered, for example, in a single dose to multiple doses per week, although in one or more embodiments, the method may include a course of treatment that includes administering a dose of the MTO-derived material at a frequency outside this range. When a course of treatment includes administering multiple doses within a particular time period, each dose may be the same or different. For example, a course of treatment may include a loading dose of an initial dose, followed by a maintenance dose that is lower than the loading dose. Also, when multiple doses are administered within a particular time period, the intervals between doses may be the same or different.

[0113] In one or more embodiments, the MTO-derived material can be administered from once a week to as a one-off dose, although in one or more embodiments, the method may be practiced by administering the MTO-derived material at a frequency outside of this range.

[0114] Thus, in one or more embodiments, the MTO-derived material may be administered minimally at least once a week, at least once a month, at least once a year, at least once every two years, at least once every three years, at least once every five years, at least once every ten years, or as a one-time administration.

[0115] In one or more embodiments, the MTO-derived material may be administered at a maximum frequency of no more than once every 5 years, no more than once every 3 years, no more than once every 2 years, no more than once a year, no more than once a month, or no more than once a week.

[0116] In one or more embodiments, the MTO-derived material can be administered at a frequency characterized by a range having endpoints defined by any minimum frequency identified above and any maximum frequency identified above that is more frequent than the selected minimum frequency. For example, in one or more embodiments, the MTO-derived material can be administered at a frequency ranging from a one-time administration to once a week, once every three years to once a week, once every five years to once a month, etc.

[0117] In certain embodiments, the MTO-derived material can be administered at a frequency equal to any minimum or any maximum frequency listed above. Thus, for example, the MTO-derived material can be administered as a one-time administration, or once every three years, once every five years, etc.

[0118] In one or more embodiments, the course of treatment can have a duration from a single administration to the remaining life of the subject. Thus, in one or more embodiments, the course of treatment with the MTO-derived material can have a minimum duration of a one-time administration, at least six months, at least one year, at least three years, at least five years, or until complete recovery.

[0119] In one or more embodiments, the course of treatment with the MTO-derived material can have a maximum duration of the remaining life of the subject, 10 years or less, 5 years or less, 3 years or less, 1 year or less, 6 months or less, or 3 months or less.

[0120] In one or more embodiments, the duration of the treatment course can be characterized by a range having endpoints defined by any minimum duration identified above and any maximum duration identified above that is greater than the selected minimum duration. For example, in one or more embodiments, the duration of the treatment course can be from a one-time administration to the remaining life of the subject, from 6 months to 5 years, from 3 months to 3 years, etc.

[0121] In certain embodiments, the duration of the treatment course may be equal to any minimum duration or any maximum duration recited above. Thus, for example, the duration of the treatment course may be a one-time administration, six months, three years, until complete recovery, or for the remaining life of the subject.

[0122] The pharmaceutical compositions described above can be used to treat diseases treatable using MTO-derived substances. Treatment of a disease can be prophylactic or can be initiated after a subject exhibits one or more symptoms or clinical signs of a disease. As used herein, the term "symptom" refers to any subjective evidence of a disease or a patient's condition. As used herein, the term "sign" or "clinical sign" refers to an objective physical observation of a particular disease that can be recognized by a person other than the patient.

[0123] Preventive treatment (e.g., treatment that is initiated before a subject develops symptoms or clinical signs of disease, such as while the infection remains subclinical) is referred to herein as treatment of a subject "at risk" of having a disease. As used herein, the term "at risk" refers to a subject who may or may not actually have the risk described. Thus, for example, a subject "at risk" of having a disease is one who has one or more risk factors associated with the disease, such as, for example, genetic predisposition, ancestry, age, sex, geographic location, lifestyle, or medical history. Treatment may be continued after symptoms have disappeared, for example to prevent or delay recurrence.

[0124] Thus, the composition can be administered before, during, or after the subject first exhibits symptoms or clinical signs of the disease. Treatment initiated before the subject first exhibits symptoms or clinical signs associated with the disease may result in a reduced likelihood that the subject will experience clinical evidence of the disease, a reduced severity of the symptoms and / or clinical signs of the disease, and / or a complete recovery from the disease, compared to subjects to whom the composition is not administered. Treatment initiated after the subject first exhibits symptoms or clinical signs associated with the disease may result in a reduced severity of the symptoms and / or clinical signs of the disease, and / or a complete recovery from the disease, compared to subjects to whom the composition is not administered.

[0125] Thus, the method involves administering an effective amount of an MTO-derived material to a subject having or at risk of having a disease treatable using the MTO-derived material. In this embodiment, an "effective amount" is an amount effective to reduce, prevent progression, ameliorate, or reverse to any extent a symptom or clinical sign associated with the disease.

[0126] disease The methods and MTO-derived products described herein can be used to treat any condition treatable using MTO-derived materials. In one or more embodiments, the condition can be a condition treatable using chondrogenic MTO-derived materials (e.g., chondrocytes and / or chondrospheres). Thus, the methods and MTO-derived materials described herein can be effective in treating any condition involving degeneration of articular cartilage. Thus, exemplary conditions include osteoarthritis of any joint (including, for example, fingers, wrists, elbows, shoulders, hips, knees, ankles, toes, temporomandibular joints, etc.), cartilage damage, intervertebral disc disease, rheumatoid arthritis, hemochromatosis, psoriatic arthritis, gout, axial spondyloarthritis, juvenile idiopathic arthritis, Saldino achondrogenesis, hypochondroplasia, lethal spondylodysplasia, Torrance congenital spondyloepiphyseal dysplasia, Kniest osteopathic dysplasia, SED with shortened metatarsals. , Czech dysplasia, spondylodistal dysplasia, spondyloepiphyseal dysplasia (SEMD), Strudwick type, Stickler syndrome type 1, mild SED with early onset arthropathy, osteochondritis dissecans, relapsing polychondritis, chondrocalcinosis, osteochondroma, enchondroma, periosteal chondroma, multiple chondromatoses, enchondromatosis, chondroblastoma, chondromyxoid fibroma, systemic lupus erythematosus, seronegative spondyloarthritis, or temporomandibular joint disorders.

[0127] Chondrogenic MTO-derived material may be administered to a subject in an amount effective to, for example, promote regeneration of hyaline cartilage, promote production of type II collagen, reduce the presence of osteophytes, reduce the severity and / or extent of a symptom or clinical sign of a disorder involving degeneration of articular cartilage (e.g., reduce inflammation, reduce pain, reduce swelling), or any combination of two or more of the foregoing.

[0128] In one or more embodiments, the disease may be a disease treatable using neuronal MTO-derived material. Thus, the methods and MTO-derived materials described herein may be effective in treating any disease involving degeneration of neuronal cells or tissue. Thus, exemplary diseases include, but are not limited to, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, spinal muscular atrophy, progressive supranuclear palsy, multiple system atrophy, or stroke.

[0129] The neuronal MTO-derived substance may be administered to a subject in an amount effective to, for example, promote the formation of neural rosettes in a subject, promote the formation of neural progenitor cells in a subject, increase dopaminergic neurons in a subject, increase mature astrocytes in a subject, increase oligodendrocytes in a subject, increase markers of cerebral cortex formation in a subject, promote engraftment of transplanted cells in brain tissue of a subject, increase migration of transplanted cells in brain tissue of a subject, reduce the severity and / or extent of a symptom or clinical sign of a neurological disorder, or any two or more of the foregoing.

[0130] In the foregoing description and in the claims that follow, the term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements. The terms "comprises," "comprising," and variations thereof should be construed as open ended, i.e., additional elements or steps are optional and may or may not be present. Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one. The recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0131] In the foregoing description, certain embodiments may be described in isolation for clarity. Throughout this specification, references to "one embodiment," "embodiment," "particular embodiment," or "one or more embodiments" mean that a particular feature, configuration, composition, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, the appearance of such phrases in various places throughout this specification does not necessarily refer to the same embodiment of the present disclosure. Moreover, certain features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Moreover, certain features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, a feature described in the context of one embodiment may be combined with a feature described in the context of a different embodiment, unless the features are necessarily mutually exclusive.

[0132] For any method disclosed herein that includes separate steps, the steps can be performed in any feasible order, and, if desired, any combination of two or more steps can be performed simultaneously.

[0133] As used herein, the terms "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits, in particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0134] The present invention is illustrated by the following examples, it being understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein. EXAMPLES

[0135] Example 1 Generation of hiPSC-derived multi-tissue organoids (MTO) Induced pluripotent stem cell (iPSC) lines, designated 1024 (ATCC-BYS0110, Cat.#ACS-1024) and 9-1 (Lindborg et al., 2016, Stem Cells Transl Med 5(7):970-979; Ye et al., 2013, PLoS ONE 8(1):e53764), were expanded in ESSENTIAL 8 medium (E8, Fujifilm Cellular Dynamics, Inc., Madison, WI) on vitronectin (VTN-N, Thermo Fisher Scientific, Inc., Waltham, MA). MTO induction was initiated by harvesting iPSCs using sodium citrate buffer, briefly centrifuging, and resuspending in 40 μL of hydration fluid from a Cell-Mate3D μGel 40 Kit (BRTI Life Sciences, Two Harbors, MN). The suspension was then transferred to one well of a 6-well ultra-low attachment plate (COSTAR Ultra-Low Attachment Microplates, Corning Life Sciences, Corning, NY) containing 5 mL of E8 medium and incubated for 24 h. The cells and culture medium were then transferred to a G-Rex 100 bioreactor (Wilson Wolf, New Brighton, MN) containing 25 mL of E8 medium and incubated at 37°C in 5% CO2. E8 medium (Gibco, Thermo Fisher Scientific Inc., Waltham, MA) containing 1% antibiotic-antimycotic was replaced every 3-4 days throughout the entire MTO culture.

[0136] Histology and immunohistochemistry MTOs from both cell lines were harvested at weeks 8 and 11 (both cell lines) and at week 30 (1024 only) and fixed in 10% neutral buffered formalin solution for 3.5 h at room temperature. After fixation, samples were transferred to 70% ethanol solution and then processed for routine paraffin embedding. Samples were then cut into 4 mm thick sections, deparaffinized, rehydrated, and routinely stained with hematoxylin and eosin (H&E) and Alcian blue. For immunohistochemical staining, 4 mm sections were cut, deparaffinized, rehydrated, and subsequently incubated with 3% hydrogen peroxide to suppress endogenous peroxidase activity and treated with serum-free protein blocking agent (DAKO, Glostrup, Denmark) for 15 min. Sections were then subjected to appropriate antigen retrieval methods (if required) and incubated with primary antibodies for 60 min at room temperature. Color development was performed using the EnVision FLEX DAB+ substrate / developer system (Cat.# GV825, Agilent-Dako, Santa Clara, CA). Stained sections were examined under an Olympus BH-2 microscope (Olympus America, Center Valley, PA) and imaged with a SPOT Insight 4 MegaSample digital camera and SPOT Advanced software (Diagnostic Instruments Inc., Sterling Heights, MI).

[0137] Morphometry Aggrecan immunohistochemically stained tissue sections of MTO biological replicates at 8 weeks (n=7) and 11 weeks (n=5) were analyzed for percent aggrecan stained area (stained area / total tissue area) using a Nikon Eclipse E-800M brightfield / fluorescence / darkfield microscope equipped with a Nikon DXM1200 high-resolution digital camera. Images for histomorphometry were analyzed using ImageJ2 / Fiji software (National Institutes of Health, open source). Values ​​are reported as percent area (%) ± standard deviation.

[0138] MTO RNA-seq and data generation

[0139] MTO was dissolved in RLT buffer (Qiagen, Hilden, Germany) and RNA was isolated from cell lysates using the RNeasy Plus mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Extracted RNA was then quantified by RiboGreen RNA assay (Thermo Fisher Scientific, Waltham, MA) and quality / size analyzed by Agilent BioAnalyzer (Agilent Technologies, Santa Clara, CA). 2 × 50 bp FastQ paired-end reads for 6 samples (n = 62.4 million on average per sample) were trimmed using Trimmomatic (v0.33) enabled with the optional "-q" option. A sliding window trimming of 3 bp from the 3' end was required with a minimum Q30. Quality control of the raw sequence data for each sample was performed with FastQC. Read mapping was performed using Hisat2 (v2.1.0) using the human genome (GRCh38) as a reference. Gene quantification was performed using Feature Counts on raw read counts. Existing RNA-seq data was processed using the same pipeline.

[0140] RNA-seq data analysis Raw read counts (CPM) were used as input for differentially expressed gene (DGE) analysis with the DESeq2 package (v1.30.1) in R (v4.0.5) (Love et al., Genome Biol. 15:550 (2014)). Benjamini-Hochberg correction was used to adjust p-values ​​(p-adj). Significance was determined using a cutoff of 0.05 (FDR corrected p<0.05) and a minimum absolute fold change value of 2-fold. Ontology enrichment of the DGE results was performed using the TopGo package (v2.42.0; Alexa, A. and Rahnenfuhrer, J., topGO: Enrichment Analysis for Gene Ontology (2021)) as previously described (Carbon et al., Nucleic Acids Res. 49: D325-D334 (2021)). Enrichment results were visualized using ClusterProfiler (v3.18.1; Yu et al., OMICS 16: 284-287 (2012)). The cutoffs for p-values ​​(after applying the Benjamini-Hochberg correction) and q-values ​​were 0.05 and 1, respectively. Expression FDR < 0.05 and log2 fold greater or less than ±2 were considered significant. Genes with changes were selected and uploaded to STRING (v11) for use in functional protein association networks of putative gene products. Gene products were clustered using Euclidean distance as previously described (Szklarczyk et al., Nucleic Acids Res 47:D607-D613 (2019)). Local network clusters were downloaded from STRING analysis as previously described (Szklarczyk et al., Nucleic Acids Res 47:D607-D613 (2019)).

[0141] [Table 3]

[0142] Additional statistics Histomorphometry values ​​of MTO tissue sections were compared using unpaired t-tests. p-values ​​are reported (α=0.05). Two biological replicates for MTO were used for RNA-Seq. All gene expression data used for visualization and statistical testing were first normalized and transformed using rlog() and assay() in DESeq2 as previously described (Love et al., Genome Biol. 15:550 (2014)). Statistical results for expression changes of grouped genes were obtained using one-sided Wilcoxon signed rank tests. p-values ​​are reported (α=0.05). Expression of single genes was compared using one-sided pairwise t-tests. p-values ​​are reported (α=0.05). Confidence intervals are reported where applicable.

[0143] Example 2 Human iPSCs were induced to form organoids as described in Example 1, but using a buffered hyaluronic acid (HA) solution instead of the hydration solution from the Cell-Mate 3D μGel 40 Kit (BRTI Life Sciences, Two Harbors, MN). After culture in low-attachment plates, iPSCs were transferred to E8 medium in G-Rex 100 bioreactor flasks (Wilson Wolf, New Brighton, MN). The resulting hyaluronic acid-producing MTOs were then maintained in long-term cell culture for 12 or 30 weeks in E8 medium in G-REX 100 bioreactor flasks (GREX, Wilson Wolf Inc., New Brighton, MN).

[0144] Cartilage / chondrocytes were present in the resulting multi-tissue organoids (MTOs). Developing hyaline cartilage was evident in the hyaluronic acid-producing MTOs after 12 weeks. After 30 weeks, mature hyaline cartilage morphology was observed with chondrocytes surrounded by abundant matrix, and immunohistochemical (IHC) staining for aggrecan and type II collagen was consistent with hyaline cartilage.

[0145] Example 3 The procedure was the same as in Example 2, except that the input iPSCs were resuspended in a buffered hyaluronic acid solution and directly transferred to a G-REX 100 bioreactor for culture. Nervous and cartilage tissues were obtained by this method. FIG. 23.

[0146] Example 4 A rat model of osteoarthritis Treated joints received 2M chondrocyte-derived cells (SARCart cells, Sarcio, Inc., Minneapolis, MN) in Hank's balanced salt solution (HBSS) at the time of surgery and again 3 weeks after surgery. Control joints received HBSS alone. Osteoarthritic lesions were scored from 0 (normal) to 12 (severe) in a blinded manner by a physician. Figure 9 shows representative hematoxylin and eosin (H&E) staining of treated and untreated knee joints in one of six animals. The untreated knee showed moderate to severe osteoarthritis (scores 5–12) in the untreated control joints and no disease to mild osteoarthritis (scores 0–4) in the treated joints, indicating a significant therapeutic effect of organoid-derived chondrocytes. Antibody specific for human Ku80 (arrows) in Figure 10 confirms engraftment of human SARCart cells in the treated rat osteoarthritis model. No staining for Ku80 was seen in control joints.

[0147] Example 5 A goat model of cartilage injury repair Figures 12 and 13. Cartilage damage was surgically induced by an 8 mm diameter full-thickness injury in the non-weight-bearing part of the articular cartilage in the stifle (knee) joint of two goats. At the time of surgery, the lesions were treated as follows: In goat #1 (male), the treated side received single-cell chondrocyte product derived from MTO-derived chondrocyte spheres embedded in fibrin glue (TISSEEL, Baxter International Inc., Deerfield, IL), and the control side received fibrin glue only. In goat #2 (female), the treated side received MTO-derived chondrocyte spheres embedded in fibrin glue, and the control side received fibrin glue only. Twelve weeks after surgery, the goats were sacrificed and the knees were examined grossly pathologically, then fixed in neutral buffered formalin (NBF) and subjected to histopathological examination. Histology showed significant neocartilage formation in the treated lesions and minimal or no regeneration in the control lesions.

[0148] Example 6 Generation of chondrocytes Organoids were generated as described in Example 1 or Example 2. Organoids were harvested from the bioreactor, transferred to a 50 mL conical tube, and centrifuged at 200×g for 5 minutes. The supernatant was removed, and the pellet was resuspended in 5 mL of TRYPLE EXPRESS enzyme (Thermo Fisher Scientific, Inc., Waltham, MA) containing 500 μg of DNase 1 (Thermo Fisher Scientific, Inc., Waltham, MA). Organoids were incubated at 37° C. for 5 minutes, and then mechanically dissociated with a 5 mL serological pipette. Dissociated cells were filtered using a 100 μm cell strainer (BD Falcon, Franklin Lakes, NJ), and organoids collected on the filter were transferred to a 50 mL conical tube containing 5 mL of 0.15% collagenase type II (Stem Cell Technologies, Inc., Vancouver, British Columbia, Canada) in DMEM / F12 containing 500 μg of DNase 1 (Thermo Fisher Scientific, Inc., Waltham, MA).

[0149] The organoids were reincubated at 37 °C for 5 min and subjected to a second mechanical dissociation using a 5 mL serological pipette. The mixture was filtered through a 100 μm cell strainer and the filter was washed with 5 mL of DMEM / F12. The organoids collected on the filter were transferred to a new 50 mL conical tube containing 5 mL of 0.15% collagenase (Stem Cell Technologies, Inc., Vancouver, British Columbia, Canada) in DMEM / F12 containing 500 μg of DNase 1 (Thermo Fisher Scientific, Inc., Waltham, MA).

[0150] Organoids were re-incubated at 37 °C for 5 min and then subjected to mechanical dissociation using a p1000 pipette. Dissociated organoids were filtered through a 100 µm cell strainer, the filter was washed with 15 mL of DMEM / F12, and the flow-through was collected.

[0151] The cells from the flow-through were counted and then centrifuged at 200×g for 5 min to remove the supernatant. At this point, the organoids were completely dissociated into single cells and could be used as single cells or re-cultured in E8 medium to form chondrspheres.

[0152] To form chondrocytes, the dissociated organoid cell pellet was transferred to a G-Rex 100 bioreactor (Wilson Wolf, New Brighton, MN) containing 35 mL of E8 medium (Fujifilm Cellular Dynamics, Inc., Madison, WI) and incubated at 37 °C for approximately 2 weeks with medium changes twice a week. Chondrocytes were collected in a 50 mL conical tube and centrifuged at 200 × g for 5 min. The supernatant was removed and the pellet was suspended in 1 mL of TRYPLE EXPRESS enzyme (Thermo Fisher Scientific, Inc., Waltham, MA) containing 100 μg of DNase 1 (Thermo Fisher Scientific, Inc., Waltham, MA) and then incubated at 37 °C for 5 min.

[0153] Chondrspheres were manually dissociated five times using a p1000 pipette and then filtered through a 100 μm cell strainer, which was then washed with at least 9 mL of DMEM / F12. If chondrspheres remained on the filter, they were collected and transferred to a new 50 mL conical tube containing 1 mL of TRYPLE EXPRESS enzyme (Thermo Fisher Scientific, Inc., Waltham, MA) with 100 μg of DNase 1 (Thermo Fisher Scientific, Inc., Waltham, MA) and then incubated at 37 °C for 5 min. The chondrspheres were manually dissociated five times using a p1000 pipette and then filtered through a 100 μm cell strainer. The cell strainer was then washed with at least 9 mL of DMEM / F12.

[0154] The filtered cells were counted and incubated on ice. The cells were centrifuged at 200×g for 5 min. The supernatant was removed and the pellet was resuspended in E8 medium and aliquoted into multiple tubes according to the test parameters and the intended dose per animal. For injection into animals, the chondrocytes were washed once with ice-cold Hank's Balanced Salt Solution (HBSS), centrifuged at 200×g for 5 min, and then resuspended in HBSS. Rats were administered 2M cells suspended in 50 μL HBSS by intra-articular injection. Figures 9 and 10.

[0155] Example 7 Neuronal MTO-derived material was generated by washing iPSCs three times with 17.5 mL of PBS, then adding 17.5 mL of passaging solution / citrate buffer to the flask containing the washed iPSCs. The culture was observed for 5 minutes or until the cells began to lift off, at which point the passaging solution / citrate buffer was aspirated. The cells were then washed with 10 mL of DMEM / F12 (Thermo Fisher Scientific, Inc., Waltham, MA) and collected in a 50 mL conical tube (total volume of 30 mL). The cells were centrifuged at 150×g / 1200 RPM for 5 minutes. The supernatant was aspirated and the cell pellet was resuspended in 250 μl of CELL-MATE3D hydrating fluid (BRTI Life Sciences, Two Harbors, MN) and mixed by vortexing according to the manufacturer's protocol. The CELL-MATE3D matrix was transferred to a funnel apparatus and centrifuged to 2700 RPM according to the manufacturer's protocol.

[0156] Small pieces (10 μl-30 μl) of cell-loaded CELLMATE 3D matrix were excised using a scalpel and added to a G-REX 100 cell culture device (Wilson Wolf Corporation, St. Paul, MN) containing 50 mL of ESSENTIAL 8 medium (Thermo Fisher Scientific, Inc., Waltham, MA). Cells were incubated in the G-REX 100 cell culture device at 37 °C (5% CO2, 20% O2) for 14-28 days, with the culture medium changed every 3-4 days. Characteristics of the resulting organoids are shown in Figures 13-17.

[0157] Example 8 MTO-derived neural tissue was prepared by embedding 20 μM iPSCs in Cell-Mate 3D microgels and culturing them in E8 medium according to the manufacturer's instructions. Cells were incubated at 37°C (5% CO2, 20% O2) in a G-REX 100 cell culture apparatus (Wilson Wolf Corporation, St. Paul, MN) for 8.5–14 weeks (Figure 18).

[0158] Example 9 MTO-derived neural tissue prepared as described in Example 8 was used in this example.

[0159] animal Adult female nude rats (rnu / rnu; 210 g ± 20 g; n = 5) were purchased from Taconic biosciences (Rensselaer, NY) and transplanted with 6-week-old organoid-derived cells for the treatment of induced hemiparkinsonism.

[0160] Induction of hemiparkinsonism by 6-OHDA Rats were anesthetized with vaporized isoflurane (Piramal Healthcare, Mumbai, India) and placed in a stereotaxic frame (David Kopf Instruments, Inc., Tujunga, CA). The heads of the rats were shaved, treated with iodine solution, and ophthalmic gel was applied to the eyes (VETERICYN PLUS, Innovacyn, Inc., Rialto, CA). A single midline incision was made along the scalp and the skin was retracted to expose the bregma. A 10 μl Hamilton syringe (Hamilton Co., Reno, NV) was filled with 3 μg / μl 6-hydroxydopamine hydrochloride (6-OHDA; Millipore-Sigma; St. Louis, MO) suspended in 1 mg / mL ascorbic acid solution (Hospira, Inc., Lake Forest, IL) in 0.9% NaCl. A small burr hole was drilled in the skull above the injection site in the right hemisphere (from bregma: 4.4 mm posterior, 1.2 mm lateral). The needle was slowly inserted into the brain 7.6 mm ventral to the pia mater and 2.2 μl of 6-OHDA solution was injected at a rate of 0.5 μl / min. After injection, the needle was left in place for 2 minutes and then slowly withdrawn. A second burr hole was then drilled in the skull above the second injection site in the right hemisphere (from bregma: 4.0 mm posterior, 1.4 mm lateral). The needle was slowly inserted into the brain 7.8 mm ventral to the pia mater and 1.8 μl of 6-OHDA solution was injected at a rate of 0.5 μl / min. After completion of the injection, the needle was left in place for 2 minutes and then slowly withdrawn. The incision site was cleaned and closed using a wound stapler (AUTOCLIP, Fine Science Tools, Foster City, CA). Rats were then placed in a heated recovery cage until fully sternal. Buprenorphine-SR (1 mg / kg; ZooPharm, Windsor, CO) was administered subcutaneously at the time of surgery.

[0161] In vivo assessment of treatment response by rotational analysis Rats were tested for rotational bias 10 and 28 days after 6-OHDA lesions and every 2 weeks after organoid transplantation. Rats were placed in a clear plastic cylinder (38 cm diameter × 34.5 cm height) under a ceiling-mounted video camera. After an initial 10-min habituation period, 0.9% NaCl was injected intraperitoneally and recorded for 20 min. Rats were then injected intraperitoneally with 5 mg / kg D-amphetamine (Millipore-Sigma, Burlington, MA) in 0.9% NaCl and recorded for 40 min. Recordings were analyzed using Fiji imaging software (Schindelin et al., 2012, Nature Methods 9:676-682) with code written by staff at the University of Minnesota Imaging Center. Automated counts were confirmed in a subset of animals by visual counts. For each animal, the number of clockwise and counterclockwise rotations was counted and calculated as the mean number of rotations per minute. Animals with a mean D-amphetamine rotation score of 7 clockwise rotations per minute were included in the implantation study. All post-implant rotation scores are reported as a percentage of rotations relative to baseline (28 days post-lesion).

[0162] Preparation of organoids for transplantation Before transplantation, the midbrain organoids were dissociated into a single cell suspension. Briefly, the organoids were rinsed in PBS and then treated with 2 mL of 0.05% trypsin-EDTA (Life Technologies, Inc., Carlsbad, CA) at 37°C for 2 minutes. 2 mL of Trypsin-EDTA supplemented with 200 μg DNase1 (Millipore-Sigma, Burlington, MA) was further added and mechanically dissociated using a p1000 pipette. The organoids were then incubated at 37°C for 5 minutes, after which the cells were again mechanically dissociated and cold Hank's balanced salt solution was added to a final volume of 10 mL. The cells were centrifuged at 250×g for 5 minutes at 4°C. The resulting supernatant was removed and the cell pellet was resuspended in 10 mL of cold HBSS and passed through a 70 μm nylon cell strainer (BD Biosciences, San Jose, CA). The cells were centrifuged a second time and the resulting pellet was resuspended in 1 mL of cold HBSS and counted using a cell counting chamber. The cells were centrifuged a third time and counted at approximately 5 × 10 cells per μl of cold HBSS. 4 The cells were resuspended at a concentration of 100 mM NaCl. The final cell solution was counted and viability was assessed using trypan blue exclusion. The final cell number was calculated as the total number of viable cells per μl.

[0163] transplant The rat's head was shaved and treated with betadine. A single midline incision was made along the scalp and the skin was retracted to expose the bregma. A 10 μl Hamilton syringe (Hamilton Co., Reno, NV) was filled with cell solution. A small burr hole was drilled in the skull above the injection site in the right hemisphere (from bregma: 1.0 mm anterior, 3.0 mm lateral). The needle was gently inserted into the brain 6.5 mm ventral to the pia mater and 1 × 10 5 Viable cells were injected at a rate of 0.5 μl / min. After injection, the needle was left in place for 1 min. Injections were repeated 5.5 mm and 4.5 mm ventral to the pia mater, with 1 × 10 cells injected at each site. 5 Inject a total of 3 x 10 cells. 5viable cells were injected. After the last injection, the needle was left in place for 3 minutes and then slowly withdrawn. The wound site was cleaned and closed using a wound stapler (AUTOCLIP, Fine Science Tools, Foster City, CA).

[0164] Tissue collection Eight weeks after implantation, rats were deeply anesthetized using vaporized isoflurane. Rats were perfused transcardially with ice-cold PBS followed by ice-cold 4% paraformaldehyde fixative. Brains were removed and immersed in fixative overnight at 4°C. Fixed tissues were routinely processed for paraffin embedding, cut into 4 μm thick sections, deparaffinized, rehydrated, stained with hematoxylin and eosin, and immunohistochemically stained for STEM121 antigen to demonstrate human cells.

[0165] The complete disclosures of all patents, patent applications, and publications, and electronically available materials (including, for example, nucleotide sequence entries in GenBank and RefSeq, amino acid sequence entries in, for example, SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety when cited herein. In the event of any inconsistency between the disclosure of this application and the disclosure in any document incorporated herein by reference, the disclosure of this application shall prevail. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be understood from those descriptions and examples. The invention is not limited to the exact details shown and described, and variations obvious to one skilled in the art will be included in the invention defined by the claims.

[0166] Unless otherwise indicated, all numerical values ​​expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be understood in all instances to be modified by the term "about." Accordingly, unless specifically indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0167] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviation found in their respective testing measurements. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so stated.

Claims

1. 1. A method for producing multi-tissue organoids (MTO), comprising: Obtaining pluripotent stem cells (PSCs); and inducing the growth of multi-tissue organoids by culturing the harvested PSCs in suspension in a culture vessel.

2. 1. A method for producing multi-tissue organoids (MTO), comprising: introducing pluripotent stem cells into a cell culture medium containing hyaluronic acid; transferring the pluripotent stem cells to a three-dimensional matrix-free cell culture device; Culturing the pluripotent stem cells in the cell culture device for at least one week; and generating a multi-tissue organoid comprising cartilage, bone, fibrous connective tissue, brain tissue, or epithelial tissue, or a combination thereof.

3. The cell culture medium contains hyaluronic acid, fibroblast growth factor, transforming growth factor beta (TGFβ), growth differentiation factor 5 (GDF-5), DMEM / F12, magnesium L-ascorbic acid-2-phosphate, sodium selenium, insulin, NaHCO ３ 10. The method of claim 1, further comprising one or more of: transferrin, TGFβ1, NODAL, or bone morphogenetic protein 2 (BMP-2).

4. 4. The method of claim 2 or 3, wherein the cell culture device comprises a second cell culture medium.

5. The method of any one of claims 1 to 3, wherein culturing the cells in the cell culture device comprises culturing the cells at room temperature.

6. The method of any one of claims 1 to 3, wherein culturing the cells in the cell culture device comprises culturing the cells at 37°C.

7. The method of any one of claims 1 to 3, wherein the PSCs are incubated in a bioreactor.

8. The method of any one of claims 1 to 3, wherein the organoid comprises cartilage, bone, fibrous connective tissue, or a combination thereof.

9. The method of claim 8 , wherein the cartilage comprises hyaline cartilage.

10. The method of any one of claims 1 to 3, wherein the organoid comprises nerve cells or nerve tissue.

11. The method of any one of claims 1 to 3, further comprising isolating the organoid.

12. 4. The method of any one of claims 1 to 3, further comprising disaggregating the cells of the organoid to generate an individualized population of cells.

13. 13. The method of claim 12, further comprising culturing cells from said population of individualized cells.

14. The method of claim 13 , wherein the cells comprise chondrocytes.

15. 10. The method of claim 9, further comprising isolating chondrocytes from the organoids.

16. The method of claim 15, further comprising forming a chondrocyte aggregate.

17. 16. The method of claim 15, further comprising culturing the chondrocytes on an ultra-low attachment surface or in chondrogenic medium, or both.

18. 16. The method of claim 15, further comprising culturing the chondrocytes in a bioreactor.

19. The organoids are cells expressing transforming growth factor β1 (TGFβ1); Cells expressing fibroblast growth factor 2 (FGF2); cells expressing bone morphogenetic protein 2 (BMP2); cells expressing bone morphogenetic protein 6 (BMP6); cells expressing growth differentiation factor 5 (GDF5); Cells expressing secreted frizzled-related protein 1 (SFRP1); Cells expressing inhibin subunit βA (INHβA); cells expressing transforming growth factor beta 3 (TGFβ3); cells expressing insulin-like growth factor 2 (IGF2); cells expressing leukemia inhibitory factor (LIF); cells expressing bone morphogenetic protein 4 (BMP4); cells expressing BMP endothelial cell precursor-derived regulator (BMPER); Cells expressing left-right determining factor 1 (LEFTY1); or The method according to any one of claims 1 to 3, comprising a combination thereof.

20. The method according to any one of claims 1 to 3, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

21. The method according to any one of claims 1 to 3, wherein the culture vessel or cell culture device is free of a biomimetic coating.

22. A pharmaceutical composition for use in a method for treating a subject having or at risk of having a disease involving degeneration of articular cartilage, comprising administering to the subject a composition comprising a chondrogenic MTO-derived material in an amount effective to improve at least one symptom or clinical sign of the disease.

23. 23. The pharmaceutical composition of claim 22, wherein the MTO-derived material comprises chondrocytes, chondrocytes, or both.

24. 24. The pharmaceutical composition of claim 22 or claim 23, wherein the chondrogenic MTO-derived material is administered in an amount effective to promote hyaline cartilage regeneration, promote type II collagen production, reduce the presence of osteophytes, reduce joint pain, reduce joint inflammation, or any combination of two or more of the foregoing.

25. The disease is osteoarthritis, cartilage damage, intervertebral disc disease, rheumatoid arthritis, hemochromatosis, psoriatic arthritis, gout, axial spondyloarthritis or juvenile arthritis, Saldino-type achondrogenesis, hypochondroplasia, lethal planovaginal dysplasia, Torrance-type congenital spondyloepiphyseal dysplasia, Kniest dysplasia, SED with shortened metatarsals, Czech dysplasia, spondyloperipheral dysplasia, spondyloepiphyseal dysplasia (SEMD), The pharmaceutical composition according to claim 22 or 23, which is for Strudwick syndrome, Stickler syndrome type 1, mild SED with early-onset arthropathy, osteochondritis dissecans, relapsing polychondritis, chondrocalcinosis, osteochondroma, enchondroma, periosteal chondroma, multiple chondromatoses, enchondromatosis, chondroblastoma, chondromyxofibroma, rheumatoid arthritis, juvenile idiopathic arthritis, gout, systemic lupus erythematosus, seronegative spondyloarthritis, or temporomandibular joint disorder.

26. A pharmaceutical composition for use in a method for treating a subject having or at risk of having a disease involving degeneration of nerve cells or nerve tissue, comprising administering to the subject a composition comprising a nerve cell MTO-derived substance in an amount effective to ameliorate at least one symptom or clinical sign of the disease.

27. 27. The pharmaceutical composition of claim 26, wherein the neuronal MTO-derived substance is administered in an amount effective to promote neural rosette formation in the subject, promote the formation of neural progenitor cells in the subject, increase dopaminergic neurons in the subject, increase mature astrocytes in the subject, increase oligodendrocytes in the subject, increase markers of cerebral corticogenesis in the subject, promote engraftment of transplanted cells within the brain tissue of the subject, increase migration of transplanted cells within the brain tissue of the subject, reduce the severity and / or degree of symptoms or clinical signs of a neurological disorder, or any two or more of the foregoing.

28. 28. The pharmaceutical composition of claim 26 or claim 27, wherein the disease is Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, spinal muscular atrophy, progressive supranuclear palsy, multiple system atrophy, or stroke.

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