Patent Application
20240279608
The disclosure provides for production processes for directed differentiation of stem cells into chondrocytes in fully defined animal free conditions, and uses thereof, including for focal repair of articular cartilage.
Degeneration of articular cartilage also known as osteoarthritis is a major cause of disability in the United States affecting more than 20 Million people. There are no efficient therapies for this condition, which explains a very high percentage of patients undergoing highly invasive and expensive total joint replacement procedure. Some novel treatments using allogenic cartilage from pediatric donors (postmortem) have recently showed promising results, but supply of this donor tissue is highly restricted, expensive and carries some risk of infection.
Osteoarthritis (OA) impacts hundreds of millions of people worldwide, with those affected incurring significant physical and financial burdens. Injury to the articular surface is a major contributing risk factor for the development of OA. Current cartilage repair strategies are moderately effective at reducing pain but often replace damaged tissue with biomechanically inferior fibrocartilage. Described herein are production processes for directed differentiation of pluripotent stem cells into chondrocytes in fully defined animal-free conditions. The chondrocytes made by the processes of the disclosure exhibited long-term functional repair of porcine articular cartilage. Accordingly, the processes disclosed herein provide a new clinical paradigm for articular cartilage repair and mitigation of the associated risk of OA.
In a particular embodiment, the disclosure provides a process to produce highly viable, engraftable human chondrocytes from pluripotent stem cells, comprising: culturing stem cells on laminin coated tissue culture vessel (s) comprising a serum-free, stabilized cell culture medium for human embryonic stem (ES) or induced pluripotent stem cells (iPSCs); transferring the stem cells to a 3-D condition/spin culture system and culturing the stem cells in a medium for 5 days or more, wherein the medium comprises a serum-free, stabilized cell culture medium for human embryonic stem (ES) or induced pluripotent stem cells (iPSCs), and a ROCK inhibitor; differentiating the stem cells to chondrocyte-like cells by culturing the stem cells under the following conditions: (a) culturing the stem cells in a mesoderm induction differentiation medium for 3 days or more, wherein the mesoderm induction differentiation medium comprises a chemically defined, serum-free hematopoietic cell medium, ROCK inhibitor, Wnt3a, Activin A, and FGF2; (b) culturing the stem cells in a MI-B day differentiation medium for 3 days or more, wherein the MI-B day differentiation medium comprises a chemically defined, serum-free hematopoietic cell medium, Wnt3a, Noggin, and FGF2; (c) culturing the stem cells in chondrogenic induction differentiation medium A for 3 days or more, wherein the chondrogenic differentiation medium comprises a chemically defined, serum-free hematopoietic cell medium, FGF2, and BMP4; sorting the differentiated stem cells by using magnetic cell separation to identify CD309 and CD326 negative cells, which are collected; culturing the CD309 and CD326 negative cells in chondrogenic induction differentiation medium B for seven days or more to form chondrocytes, wherein chondrogenic induction differentiation medium B comprises a chemically defined, serum-free hematopoietic cell medium, FGF2, IGF1, SHH, BMP4, ROCK inhibitor, and Primocin; culturing the chondrocytes in maintenance media for 7 days or more; wherein the maintenance media comprises a chemically defined, serum-free hematopoietic cell medium, FGF2, IGF1, LIF, TGF-β1, BMP4, ROCK and Primocin; optionally, treating the chondrocytes with an agent or compound that enhances anabolism, extracellular matrix secretion, promotes cell survival and or proliferation, reduces catabolismand/or reduces degeneration of extracellular matrix in chondrocytes; and optionally, cryopreserving the chondrocytes with collagen membranes in a defined, serum-free, and animal component-free freezing medium that comprises a ROCK inhibitor. In a further embodiment, the stem cells are human embryonic stem cells. In yet a further embodiment, the stem cells are ESI-017 GMP grade cells. In another embodiment, the tissue culture vessel is tissue culture flask (s) or CellSTACK® cull chambers. In yet another embodiment, the serum-free, stabilized cell culture medium for human embryonic stem (ES) or induced pluripotent stem cells (iPSCs) is mTeSR medium. In a further embodiment, the ROCK inhibitor is Y-27632. In yet a further embodiment, a billion or more stems cells are transferred to the 3-D conditions/spin culture conditions. In another embodiment, the a chemically defined, serum-free hematopoietic cell medium is X-Vivo medium. In yet another embodiment, the magnetic cell separation utilizes antibodies to CD326 and CD309 in a biotin/streptavidin pulldown system. In a certain embodiment, the chondrocytes are treated with an agent or compound that enhances anabolism, extracellular matrix secretion, promotes cell survival and or proliferation, reduces catabolism and/or reduces degeneration of extracellular matrix in chondrocytes, having the general structure of Formula I, II or III:
wherein,
X1 is S, CH, or NH; X2 is S, CH, or N; X3 is CR2 or S; Y1 is CR7 or N; Y2 is CR6 or N; Y3 is CR5 or N; Z1 is O or CH; Z2 is O, N, NH or CH; Z3 is CR9 or N; Z4 is CR8 or N; Z5 is N or CR14; Z6 is N or CR13; Z7 is N or CR12; Z8 is N or CR11; Z9 is N or CR10; v is 0 or 1; R1, R2, R8 and R9 are independently selected from H, D, (C1-C3) alkyl, (C1-C3) alkenyl, halo, cyano, hydroxyl, nitro, thiol, and amino; R3-R7 and R10-R14 are independently selected from H, D, (C1-C3) alkyl, (C1-C3) alkenyl, halo, cyano, hydroxyl, nitro, thiol, amino, OC(R15)3, OCH(R15)2, OCH2(R15),
wherein n is an integer from 1 to 5; and R15 is independently H, halo, or a (C1-C3) alkyl; and wherein the compound modulates (i) inflammatory response and/or (ii) tissue degeneration. In a further embodiment, X is selected from the group consisting of
and wherein R2 is an H, D, (C1-C3) alkyl, (C1-C3) alkenyl, halo, cyano, hydroxyl, nitro, thiol, or amino. In yet a further embodiment, Y is selected from the group consisting of:
and wherein v is 0 or 1; R3-R7 are independently selected from H, D, (C1-C3) alkyl, (C1-C3)alkenyl, halo, cyano, hydroxyl, nitro, thiol, amino, OC(R15)3, OCH(R15)2, OCH2(R15),
and wherein n is an integer from 1 to 5. In another embodiment, z is selected from the group consisting of:
and wherein, R8 and R9 are independently selected from H, D, (C1-C3) alkyl, (C1-C3) alkenyl, halo, cyano, hydroxyl, nitro, thiol, and amino; R10-R14 are independently selected from H, D, (C1-C3) alkyl, (C1-C3) alkenyl, halo, cyano, hydroxyl, nitro, thiol, amino, OC (R15)3, OCH (R15)2, OCH2(R15),
wherein n is an integer from 1 to 5; and wherein R15 is independently H, halo, or a (C1-C3) alkyl. In yet another embodiment, the compound has a structure selected from the group consisting of:
In a particular embodiment, the compound is selected from the group consisting of:
In a further embodiment, the compound interacts with (i) domain 2 of gp130 and which locks gp130 into a non-permissive conformation, (ii) produce atypical gp130 homodimers and/or (iii) modulates STAT3 and/or MYC signaling.
In a particular embodiment, the disclosure further provides for engrafting chondrocytes made by a method disclosed herein to a subject in need thereof. In a further embodiment, the chondrocytes engrafted are derived from iPSCS made from the subject's cells.
As used herein and in the appended claims, the singular forms “a,” “an, ” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a growth factor” includes a plurality of such growth factors and reference to “the stem cell” includes reference to one or more stem cells and equivalents thereof known to those skilled in the art, and so forth.
Also, the use of “or” means “and/or” unless stated otherwise. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes, ” and “including” are interchangeable and not intended to be limiting.
It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although many methods and reagents are similar or equivalent to those described herein, the exemplary methods and materials are disclosed herein.
All publications mentioned herein are incorporated herein by reference in full for the purpose of describing and disclosing the methodologies, which might be used in connection with the description herein. Moreover, with respect to any term that is presented in one or more publications that is similar to, or identical with, a term that has been expressly defined in this disclosure, the definition of the term as expressly provided in this disclosure will control in all respects.
It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.
Any specific values should be understood to mean that they are within reasonable error for time, temperature, range, etc. For example, reference to “2 minutes” should be construed as being preceded by “about”, wherein the value has reasonable error for processing and the like. In another embodiment, the term “about” includes a range of 1%-5%±the defined value.
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used to described the present invention, in connection with percentages means ±1%-5%.
As used herein, the term “collagen, ” can mean, but is in no way limited to, any of a family of extracellular, closely related proteins occurring as a major component of connective tissue, giving it strength and flexibility. At least 14 types exist, each composed of tropocollagen units that share a common triple-helical shape but that vary somewhat in composition between types, with the types being localized to different tissues, stages, or functions. In some types, including the most common, Type I, the tropocollagen rods associate to form fibrils or fibers; in other types the rods are not fibrillar, but are associated with fibrillar collagens, while in others they form nonfibrillar, nonperiodic, but structured networks. Cartilage can contain chondrocytes or chondrocyte-like cells and intracellular material, proteoglycans, and other proteins. Cartilage includes articular and non-articular cartilage.
“Articular cartilage, ” also referred to as hyaline cartilage, refers to an avascular, non-mineralized connective tissue, which covers the articulating surfaces of bones in joints and serves as a friction reducing interface between two opposing bone surfaces. Articular cartilage allows movement in joints without direct bone-to-bone contact. The cartilage surface appears smooth and pearly macroscopically, and is finely granular under high power magnification. Articular cartilage is associated with the presence of Type II and Type IX collagen and various well-characterized proteoglycans, and with the absence of Type X collagen, which is associated with endochondral bone formation.
The term “cell” can mean, but is in no way limited to, its usual biological sense, and does not refer to an entire multicellular organism. The cell can, for example, be in vivo, in vitro or ex vivo, e.g., in cell culture, or present in a multicellular organism.
As used herein, the term “stem cells, ” can mean, but is in no way limited to, undifferentiated cells having high proliferative potential with the ability to self-renew that may generate daughter cells that may undergo terminal differentiation into more than one distinct cell phenotype. These cells may be able to differentiate into various cells types and thus promote the regeneration or repair of a diseased or damaged tissue of interest in vivo, in vitro or ex vivo. The term “cellular differentiation” refers to the process by which cells acquire a cell type. The term “progenitor cell” as used herein refers to an isolatable cell of any lineage that maintains the plasticity to differentiate into one or more target cell type that includes, but is not limited to, chondrocytes, osteocytes, and adipocytes. A progenitor cell, like a stem cell, may be able to differentiate into a specific type of cell, but is already more specific than a stem cell, and is pushed, or stimulated, to differentiate into its “target” cell type. Generally, stem cells can replicate indefinitely, whereas progenitor cells can only divide a limited number of times.
As used herein, the terms “osteoprogenitor cells”, “chondroprogenitor cells”, “osteochondroprogenitor cells”, “mesenchymal cells”, “mesenchymal stem cells (MSC)”, or “marrow stromal cells” are used interchangeably to refer to multipotent stem cells that can differentiate along one or several lineage pathways into osteoblasts, chondrocytes, myocytes, adipocytes, and tendocytes.
As used herein, the term “chondrocytes” can mean, but is in no way limited to, cells found in cartilage that produce and maintain the cartilaginous matrix. The term “chondrogenesis” refers to the formation of new cartilage from cartilage forming or chondrocompetent cells.
The terms “patient”, “subject” and “individual” are used interchangeably herein, and refer to an animal, particularly a human, to whom treatment including prophylaxis treatment is provided. This includes human and non-human animals. The term “non-human animals” and “non-human mammals” are used interchangeably herein includes all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles etc. In one embodiment, the subject is human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. “Mammal” refers to any animal classified as a mammal, including humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. A subject can be male or female. A subject can be a fully developed subject (e.g., an adult) or a subject undergoing the developmental process (e.g., a child, infant or fetus).
The term “treat” or “treatment” as used herein, refers to a therapeutic treatment wherein the object is to eliminate or lessen a condition or a symptom. Beneficial or desired clinical results include, but are not limited to, elimination of symptoms or a condition, alleviation of symptoms or a condition, diminishment of extent of the condition or symptom, stabilization (i.e., not worsening) of the state of the condition or symptom, and delay or slowing of progression of the condition or symptoms.
Cell therapy has been used successfully in the clinic for more than 50 years in the form of hematopoietic stem cell transplantation1. Cell therapy, however, illuminated the need for HLA-matched donors due to graft versus host disease (GVHD) encountered during allogenic transplants, in which donor lymphocytes reacted against host tissues. In the case of allogenic solid organ transplantation, immunosuppression of the host is often required for extended periods. These aforementioned limitations in the availability and compatibility of donor tissue have prompted the search for other solutions which now potentially include embryonic stem cell (ESC) and induced pluripotent stem cell-derived (iPSC) cells and tissues. The field of pluripotent stem cell (PSC)-based regenerative medicine has advanced quickly as both iPSC- and ESC-derived cells are in clinical trials5, with transplants into immunoprivileged sites such as the eye leading the way.
In the orthopedic field, reparative therapy for articular cartilage defects has classically relied on endogenous cells via the microfracture technique. In this procedure, channels are created into the bone marrow to allow mesenchymal cells with chondrogenic potential to enter the defect and generate neocartilage. More recently, autologous chondrocyte implantation (ACI) and variations thereof including MACI (matrix-associated ACI) that rely on expansion and reimplantation of chondrocytes from the patient, have been adopted. The long-term results of ACI-based procedures appear superior to microfracture, with reduced graft failure and improved patient-reported outcomes. Despite overall satisfactory outcomes, many patients still experience complications including graft integration failure, inferior quality of neocartilage (hyaline vs. fibrocartilage), donor site morbidity and osteoarthritis. Additionally, these approaches require two surgical procedures to perform the actual repair. This has fueled the search for allogeneic sources that may provide more cells with superior chondrogenic capacity without immunocompatibility issues or the need for multiple surgeries including juvenile cartilage and PSCs.
Generation of articular chondrocytes from PSCs has been challenging as most chondrogenic cells during development are fated to undergo hypertrophy and endochondral ossification rather than adopt an articular chondrocyte identity. Provided herein is the generation articular-like chondrocytes from GFP+ human pluripotent stem that can engraft, integrate into and repair osteochondral defects in subjects. Moreover, these human cells produce all layers of hyaline cartilage after 4 weeks in vivo, including a PRG430 superficial zone.
Further provided herein is methods that allow for the large-scale production of said articular-like chondrocytes. The methods disclosed herein allowed for the assessment of long-term, clinically relevant functionality of the articular-like chondrocytes. In particular, assessment was performed with Yucatan minipigs. Yucatan minipigs provide an excellent model for pre-clinical assessment of potential orthopedic therapies due to structural similarities, comparable thickness of articular cartilage and the ability to create defects of substantial volume; in addition, their size allows for cost-efficient care and observation for extended periods of time. As shown in the studies presented herein, long-term functional repair of porcine full-thickness articular cartilage defects could be repaired with hyaline cartilage from clinical grade hESC-derived articular chondrocytes that were produced using the large-scale production techniques and methods of the disclosure.
In particular, the studies presented herein demonstrate that hES-derived chondrocytes administered as a cryopreservable, membrane-embedded formulation, supported clinically relevant, long-term repair of full-thickness articular cartilage defects in pigs. At the molecular level, the membranes were found to contain no detectable residual hES cells and be populated with chondrocytes resembling both fetal and juvenile articular chondrocytes. Based on the high expression levels of SOX5/6/9 and genes associated with proliferation, it is likely that these immature articular chondrocytes mature in vivo to upregulate matrix production and assume a proper zonal identity. Importantly, the hyaline cartilage found in repaired defects had similar biomechanical properties to naive tissue, further supporting the concept that hES-derived chondrocytes can adopt adult-like properties upon transplantation and integration with surrounding cells and/or support recruitment and differentiation of chondrogenic cells into hyaline cartilage.
Further, the studies presented herein demonstrate that the hES-derived chondrocytes made by the processes disclosed herein did not induce local inflammation or immune cell infiltration despite being a xenograft in immunocompetent recipients with no immunosuppression. The results are in direct contrast to other studies that have demonstrated that xenografted articular chondrocytes in the knee can elicit a severe immune response. Thus, the processes disclosed herein represent a definite improvement when compare with similar techniques. The significant biomechanical improvement seen in porcine cartilage defects treated with human cells is very likely the result of both autocrine and paracrine mechanisms. This suggests that factors secreted by hES-derived chondrocytes can recruit endogenous cells capable of producing hyaline cartilage and support their differentiation down this path. Several candidate factors have been identified that could act in concert to achieve this result, including IGF-1, and TIMP-1 and -2.
IGF-1 is a classic anabolic factor for chondrocytes, promoting proteoglycan synthesis and reducing catabolismthis factor can also promote chondrogenesis from mesenchymal stem cells as has also been shown for FGF-6.
TIMP-1 and -2 are inhibitors of matrix metalloproteinases and have shown to be chondroprotective and prevent matrix loss downstream of the pro-inflammatory cytokine IL-1β. Following the exposure of the knee joint and creation of the defects, significant local inflammation occurs, driving production of catabolic enzymes and promoting fibrocartilage formation which may be ameliorated via the cell-mediated delivery of counteracting proteins such as TIMP-½ and IL-1RA (a decoy receptor for the pro-inflammatory cytokine IL-1α43). Together, these potential chondrogenic paracrine factors may modulate the microenvironment of the defect to promote migration and differentiation of endogenous cells into articular cartilage.
In one embodiment, the following methodology is used:
Exemplary procedure of 3D Culture: Pre-treat culture flask/plat with Y27632 (final concentration is 10 uM) for 2 hours. Aspirate mTeSR media from 2D culture flask comprising stem cells. Wash cells with appropriate volume of DPBS without Ca++/Mg++. Add 20 mL of ReLeSR and swirl the flasks to ensure entire cell surfaces are exposed to ReLeSR. Incubate for about 1 min at room temperature. Aspirate ReLeSR and leave thin layer of liquid to prevent drying of the flask/plate. Incubate flask/plate in the incubator for about 2-7 minutes. Add an appropriate volume of mTeSR to the plate/flask. Detach colonies (e.g., approximately 0.5 mm colonies, and colonies should not be disaggregated). Transfer detached cells to a spinner flask. Transfer the spinner flask to a slow speed stirrer plate in the incubator (up to 60 RPM). Culture it up to 7 days—change media every day.
Exemplary procedure of Cell Differentiation and purification: MI-A media—Add X-Vivo media to a filtration unit with 0.2 μm filter without connecting to a vacuum line. Add 10 mM Y27632 (Final conc 10 uM), 10 ug/mL Wnt3a (final conc 10 ng/ml), 10 ug/mL bFGF (final conc 10 ng/ml), and Activin A (final conc 10 ng/ml). Filter the solution. Take out a spin flask from the slow-speed stirrer for minimum of about 30 min (up to about 1 hour). Remove about 50%-70% of the mTeSR media. Transfer the rest of media to conical spin flasks while maintaining cell aggregation. Add an appropriate volume of MI-A media to the spin flask to avoid dry out. Centrifuge the conical tube for about 3 minutes at about 300 g. Aspirate supernatant. Resuspend with an appropriate volume of MI-A media and transfer to the spin flask. Wash conical tubes with appropriate volume of MI-A media and transfer to the spin flask. Transfer the spin flask to the slow speed stirrer in the incubator. Set spin speed about 45 rpm. Incubate on the slow speed stirrer in the incubator for about 3 days.
Prepare MI-B media by adding X-Vivo media to a filtration unit with 0.2 um filter without connecting to a vacuum line. Add Wnt3a (final conc 10 ng/ml), bFGF (final conc 10 ng/ml), and Noggin (final conc 50 ng/ml). Connect the vacuum line and filter it through filtration unit. Take out a spin flask from the slow-speed stirrer for minimum of about 30 min (up to about 1 hour). Remove about 50%-70% of the MI-A media. Transfer rest of media to conical spin flasks and try not to disrupt cell aggregation. Add an appropriate volume of MI-B media to the spin flask to avoid dry out. Centrifuge the conical tube for about 3 minutes at about 300 g. Aspirate supernatant. Resuspend with appropriate volume of MI-B media and transfer to the spin flask. Wash conical tubes with appropriate volume of MI-B media and transfer to the spin flask. Transfer the spin flask to the slow speed stirrer in the incubator. Set spin speed about 45 rpm. Incubate on the slow speed stirrer in the incubator for about 3 days.
Inducing chondrogenesis: Prepare CI-A media by adding X-Vivo media to a filtration unit with 0.2 um filter without connecting to a vacuum line. Add bFGF (final conc 10 ng/ml), and BMP4 (final conc 10 ng/ml). Connect the vacuum line and filter it through filtration unit. Close cap tightly and warm it up inside of clean water bath. Remove a spin flask from the slow-speed stirrer for a minimum of about 30 min (up to about 1 hour). Remove about 50%-70% of the MI media. Transfer the rest of media to conical spin flasks while not disrupting cell aggregation. Add an appropriate volume (depending upon tissue culture size) of CI-A media to the spin flask to avoid dry out. Centrifuge the conical tube for about 3 minutes at about 300 g. Aspirate supernatant. Try to avoid touching pellets. Resuspend with appropriate volume of CI-A media and transfer to the spin flask. Wash conical tubes with appropriate volume CI-A media and transfer to the spin flask. Transfer the spin flask to the slow speed stirrer in the incubator. Set spin speed about 45 rpm. Incubate on the slow speed stirrer in the incubator for about 3 days.
Prepare CI-B media: Add a X-Vivo media to a filtration unit with 0.2 um filter without connecting to a vacuum line. Add Y27632 (Final conc 10 uM), IGF-1 (final conc 10 ng/ml), bFGF (final conc 10 ng/ml), BMP4 (final conc 50 ng/ml), and SHH (final conc 25 ng/ml). Filter it through filtration unit.
Vitronectin Coating of tissue culture substrate (following a product manual from Stemcell Technologies). Thaw Vitronectin XF (Stemcell Tech; Cat#07180) and bring CellAdhere Dilution Buffer (Stemcell Tech; Cat#07183) at room temperature. Dilute Virtonectin XF in CellAdhere Dilution Buffer to reach a final concentration of 10 ug/mL. Gently mix Vitronectin XF (do not vortex). Use the diluted Vitronectin XF to coat tissue culture plate. Gently rock the tissue culture substrate back and forth to spread the Vitronectin XFM solution evenly across the surface. Incubate at room temperature for at least 1 hour before use. Do not let the Vitronectin XFM solution evaporate. Gently tilt the tissue culture substrate on to one side and allow the excess Vitronectin XF™ solution to collect. Remove the excess solution using a serological pipette or by aspiration. Ensure that the coated surface is not scratched. Wash the tissue culture substrate once using CellAdhere™ Dilution Buffer (e.g. 1 mL/well if using a 6-well plate). Aspirate wash solution and add the appropriate volume of culture medium (e.g. 2 mL/well if using a 6-well plate).
Take out a spin flask from the slow-speed stirrer for minimum 30 min (up to 1 hour). Pre-warm 1× Tryple select media. Take out the spin flask from the incubator and aspirate 50% of excessive media. Add 50 uL of 10 mM Y27632 to the spin flask. Put spin flask to the slow speed stirrer in the incubator and incubate for about 1 to 2 hours before magnetic sorting if that will take place (MACS). Take out a spin flask from the slow speed stirrer for a minimum of about 30 min (up to about 1 hour). Take out the spin flask from the incubator and aspirate excessive media (try not to aspirate aggregates). Transfer all media to a conical tube. Wash the spin flask with cold PBS and transfer to conical tube. Centrifuge for about 3 minutes at about 300 g. Aspirate supernatant (try not to touch a pellet); If cell pellet is too big, split it to couple of tubes. Wash the pellet with cold PBS and centrifuge for about 3 minutes at about 300 g. Aspirate supernatant in the conical tubes. Resuspend the pellet with an appropriate volume of pre-warmed 1× Tryple select. Incubate the conical tube in the incubator (37° C.) for about 2 minutes. Take out the tube and pipette up and down several times. Incubate the tube in the incubator for another approximate 2 minutes. Take out the tube and pipette up and down to disrupt cell aggregates to single cells. Wash with cold PBS. Centrifuge conical tube for about 3 minutes at 300 g. Carefully aspirate supernatant. Resuspend cell pellet with CI-B media. Put flask in the incubator for 24 hours. Exemplary cell density and media volumes:
Prepare collagen membrane: Take out collagen membrane. Transfer collagen membrane to sterile tissue culture substrate. Rough side should be facing up and smooth side should be facing bottom of tissue culture substrate. Pre-soak collagen membrane with CI-B media. Incubate at 37 degrees.
Seeding cells on collagen membrane: Take out tissue culture from the incubator. Removed CI-B media and wash with DPBS without Ca++and Mg++. Remove DPBS and add an appropriate volume for the tissue culture substrate of ReleSR or 1× Tryple Select. Make sure all cell surfaces are exposed to ReleSR for a minute. Remove ReleSR and incubate in 37 degrees for about 2-3 minutes. Add an appropriate volume of CI-B media to neutralize. Transfer all cells to conical tube. Count cell number using an automated hematocytometer with Trypan blue. Seed cells on the collagen membrane (5 million cells per cm2) in a dish/plate. Incubate dish/plate in hypoxia chamber for 3-4 days.
Maintenance: Prepare Maintenance media (if collagen membranes cultures in bioreactor, scale up the media proportionally): Add a X-Vivo media to a filtration unit with 0.2 um filter without connecting to a vacuum line. Add IGF-1 (final conc 10 ng/mL), bFGF (final conc 10 ng/ml), LIF (Final conc 50 ng/ml), TGFb1 (final conc 10 ng/ml), and BMP4 (final conc 1 ng/ml). Connect with the vacuum line and filter it through filtration unit. Remove dish/plate containing collagen membrane seeded with cells from the hypoxia chamber. Remove CI-B media from dish/plate (try not to touch collagen membrane and keep wet)—Change media every 2-3 days. Incubate in hypoxia chamber up to day 40 starting from Mesodermal induction (MI-A).
Cryopreserve membrane: Add appropriate volume for vial size of MesenCult ACF freezing media with 5 uL of 10 mM Y27632 in a cryo vial. Take out a plate from the hypoxia chamber Using a tweezer and carefully transfer collagen membranes to cryo vials (e.g., carefully roll them up and put them in a cryo vial). Store cryo vials in freezing container and transfer freezing container to −80 for 24 hrs. Then transfer cryo vials to liquid nitrogen tanks for longer storage.
Collagen with seeded cells can be used (or thawed and used) for cartilage repair. The method includes implanting a collagen membrane composition of the disclosure containing seeded chondrocytes to a cartilage defect site. The collagen membrane can be sized or configured to fit the defect site. The collagen membrane can be sutured or glued in place.
The following examples are intended to illustrate but not limit the disclosure. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used.
General Methods: For all experiments, independent experiments with biological replicates were employed to generate data. For in vitro experiments expected to yield large differences, standard practice of using 3 replicates was followed. All statistical methods are described in the figure legends.
Culture of pluripotent stem cells and generation of skeletal progenitors: For all experiments, research grade ESI-017 were used; the stock line was purchased from BioTime. Cells were cultured on ESC-grade Matrigel (Corning) in mTesR1 media and passaged using ReLeSR (Stemcell Technologies) according to manufacturer's instructions. Batch scale production of ESI-017 cells was carried out in CellSTACK-5 flasks, yielding ˜1 billion cells per run. Each batch was tested for mycoplasma contamination by PCR (Sigma). Cells were then transferred to suspension culture in mTesR1 for up to 5 days in spinner flasks (60 RPM). Differentiation was conducted as described in Ferguson et al. (Nature Communications 9:3634 (2018)) and Evseenko et al. (Proceedings of the National Academy of Sciences 107: 13742 (2010)). Briefly, mTesR1 was replaced with Mesoderm Induction Media A (MIM-A) for 3 days (X-Vivo containing ROCK inhibitor, FGF2, Wnt3a and Activin A) followed by MIM-B for 3 days (X-Vivo containing Wnt3a, Noggin and FGF-2). Media was then changed to Chondrogenic Induction Media A (CIM-A; X-Vivo containing BMP-4 and FGF-2) for 4 additional days. Skeletal progenitors were then isolated using MACS by depleting for CD326 and CD309; for 2 hours before starting MACS isolation, ROCK inhibitor was added to the cell culture media. The purity of cells isolated using MACS was routinely assessed using flow cytometry.
Generation of chondrospheres: Skeletal progenitors from MACS were then cultured in EZSphere plates (Nacalai USA) at 100,000 cells per well in CIM-B15 (X-Vivo containing Shh, ROCK inhibitor, BMP-4, FGF-2, IGF-1 and Primocin) in 5% oxygen for 3-5 days to form chondrogenic aggregates. After firm aggregates were verified with microscopy, they were transferred to a perfusion bioreactor (Applikon Biotech) in Maturation Media (MM; X-Vivo media containing FGF-2, BMP-4, Shh, IGF-1, LIF, TGF-31 and Primocin) until d40 of differentiation. Conditions in the bioreactor were kept at 37° C., 5% O2, 5% CO2 and 30 RPM. Fresh media was added at a rate of 1 mL/hour. Upon maturation, chondrospheres contained 5×104 cells on average. At d40, chondrospheres were cryopreserved in MesenCult-ACF plus ROCK inhibitor (Y27623, 10 μM; Tocris) and stored in liquid nitrogen until use.
Generation of hES-derived chondrocytes on membranes: Skeletal progenitors were isolated via MACS and seeded onto porcine collagen I/III membranes (Cartimaix; Matricel) sized with a 6 mm biopsy punch at two different densities designed to yield 6×105 or 3×106 million cells. Membranes were then cultured in 5% oxygen for 3-5 days in CIM-B and then transferred to the bioreactor as above for chondrospheres with the exception that rotation was not started until 3 days after transfer of membranes and was at 15 RPM. At d40, membranes were cryopreserved using MesenCult-ACF as above for chondrospheres.
Generation of hBMSCs on membranes (hBMSC-M). Human bone marrow stromal cells (n=3 donors aged 19, 68, and 87 years (all P1); pooled) were seeded onto porcine collagen I/III membranes (Cartimaix; Matricel) sized with a 6 mm biopsy punch at a total cell number of 3×106. Membranes were then cultured in 5% oxygen for 3-5 days in CIM-B, then maintained with MM until d40. At d40, membranes were digested for single cells and scRNA-sequencing was performed.
Optimization of cryopreservation media: Live cell numbers per batch of chondrospheres or membranes were determined prior to freezing using the Live/Dead Cell Viability Assay (Biovision). Chondrospheres or membranes were then cryopreserved following the manufacturer's instructions for each product (Prime XV FreezIS, Irvine Scientific; MesenCult-ACF, CryoStor CS5 or 10, mFreSR; Stemcell Technologies), thawed and then viability from the same batch compared to the starting viability before freezing.
Quantitative Real-Time PCR: Power SYBR Green (Applied Biosystems) RT-PCR amplification and detection was performed using an Applied Biosystems Step One Plus Real-Time PCR machine. The comparative Ct method for relative quantification (2-ΔΔCt) was used to quantitate gene expression. TBP (TATA-box binding protein) was used for gene normalization and expressed relative to a calibrator (sample in each set with lowest expression). Primer sequences used for qPCR are available on request. For quantification of human cell numbers in pig samples, a human TERT Taqman assay was used (Thermo). A standard curve was created with known numbers of human cells, which both determined the detection threshold as well as allowed calculation of human cell numbers in a sample based on Ct values.
Large animal model of articular cartilage repair. Yucatan minipigs were purchased from S & S farms at 6 months of age and housed under the supervision of the USC Department of Animal Resources (DAR). All preoperative, surgical and post-operative procedures were conducted following USC DAR guidelines and were overseen by the USC Institutional Animal Care and Use Committee (IACUC). Five animals per group (main study) with 2 defects each were included based on power calculations for significance. Calculation of animal numbers was based on the following statistical formula: n=1+2C(s/d)2, where C is a constant, s is expected standard deviation and d is expected difference between means. Every care was been taken to minimize the number of pigs required. Power analyses were performed for all animal studies as follows: For all treatment groups, with an alpha of 5%, and power level of 80%, N=5 per treatment group is used. Estimated standard deviation and differences between means used for calculations are based on pilot studies. Animals were anesthetized for surgery using Telazol/Xylazine 2.2-4.4 mg/kg administered intramuscularly. Medial para-patellar arthrotomy was performed by inserting microsurgical scalpel medially and proximally to the insertion of the patellar tendon on the tibia and extending it proximally until the attachment of the quadriceps muscle. The medial margin of the quadriceps was separated from the muscles of the medial compartment. The joint was extended and the patella dislocated laterally. The joint was then be fully flexed to expose the patellar groove. Two full-thickness injuries 6 mm in diameter were made using a disposable biopsy punch. No randomization was applied. For animals receiving chondrospheres in the pilot study, surgical fibrin glue (Ethicon) with or without chondrospheres was applied directly to the defect areas. For animals receiving membranes, pre-sized membranes were applied to the defect and secured with fibrin glue. Arthrotomies were then relieved, wounds sutured and animals monitored for recovery. Following 1 or 6 months, animals were sacrificed after receiving anesthesia as above using pentobarbitol (100 mg/kg).
Tissue collection and digestion: Adult human primary tissue samples were obtained from National Disease Research Interchange (NDRI). Consented, de-identified human fetal tissues were obtained from Novogenix Laboratories or the University of California Los Angeles (UCLA) Center for AIDS Research (CFAR) Gene and Cellular Therapy Core following institutional review board (IRB) approval. Articular chondrocytes from the 7-year old human juvenile subject were obtained from leftover tissues from surgical procedures approved by the UCLA institutional IRB, with patient consent and de-identification. Use of human tissues was IRB exempt by the UCLA Office of the Human Research Protection Program (IRB #15-000959). Human primary tissues or hES-derived chondrocytes on membranes were manually cut into small pieces and digested 4-16 hrs at 37° C. with mild agitation in digestion media consisting of DMEM (Corning) with 10% FBS (Sigma), 1 mg/mL dispase (Gibco), 1 mg/mL type 2 collagenase (Worthington), 10 μg/mL gentamycin (Teknova) and 100 μg/ml primocin (Invivogen).
Immunohistochemistry (IHC): Tissues were fixed in 10% formalin and sectioned at 5 μm. For DAB, sections were deparaffinized using standard procedures and antigen retrieval was performed by bringing samples to a boil in 1× citrate buffer pH 6.0 (Diagnostic Biosystems), and incubating at 60° C.for 30 minutes followed by 15 minutes cooling at room temperature. Endogenous peroxidase activity was quenched by treating samples with 3% H2O2 for 10 minutes at RT. Sections were then blocked in 2% normal horse serum for 20 minutes. Sections were then incubated with primary antibodies diluted in TBS with 1% BSA (Sigma) overnight at 4° C. Sections were washed 3 times with TBS 0.05% Tween 20 (Sigma) (TBST) before addition of HRP-conjugated secondary antibody for 30-minute incubation at RT. Sections were washed 3 times with TBST after secondary incubation and DAB substrate was then added until positive signal was observed. Sections were then immediately washed with tap water, counterstained in hematoxylin for 30 seconds and washed again with tap water before dehydration and mounting. For Hematoxylin and eosin staining, sections were deparaffinized, rinsed in tap water, and stained with Hematoxylin for 3 minutes. Sections were then washed in tap water and stained with Eosin for 2 minutes before a final wash in tap water. Safranin O/Fast Green staining was performed. To quantitate cartilage repair, the ICRS II scoring system was employed by two blinded observers. Toluidine Blue and Alcian Blue staining was performed on deparaffinized sections in accordance with standard laboratory techniques.
Biomechanical assessment of defect repair: Freshly harvested porcine cartilage tissues 6 months of age were affixed to the sample holder of the Mach-1 Mechanical Tester (Biomomentum) using instant glue (Loctite 4013) and immersed in DMEM/0.9% NaCl (1:1). The Mach-1 configuration uses a spherical indenter tool attached to a highly sensitive multiaxial load cell and automated fine motor controller, allowing for compression of the cartilage by 30% of its thickness, and live recording of resultant forces generated in all x-y-z planes. The indenter tool is then replaced with a needle which penetrates the cartilage at each point until forces comparable to underlying bone are detected, allowing for accurate thickness measurement. Altogether, the forces generated during indentation and thickness measurements are used to calculate the instantaneous modulus, which reflects the elasticity, stiffness, and resistance to compression of the tissue. Each condyle was manually mapped for testing using the Biomomentum mapping software. On average, between 40-50 points were tested on each affected condyle, with no less than 10 points directly in the defect areas, and usually about 1-3 mm apart from each other on the surface. Each control condyle was tested for an average of 15-20 points, which were spaced about 2-5 mm apart. Mapping coordinates were input into the software and indentation analysis to map instantaneous modulus was performed with the 1 mm spherical indentor tool with the following parameters: Z-contact velocity: 0.1000 mm/s; contact criteria: 0.1003 N; scanning grid: 0.2000 mm; indentation amplitude: 0.300 mm; indentation velocity: 0.300 mm/s; relaxation time: 5s. To map thickness, the indentor tool was replaced with a 26 G ¾ inch hypodermic needle and the mapping executed with the following needle penetration parameters: stage axis: position z; load cell axis: Fz; direction: positive; stage velocity: 0.2000 mm/s; contact criteria: 2.000 N; stage limit: 15 mm; stage repositioning: 2× load resolution; offset: 0. Heat maps were generated with the Mach-1 software provided by Biomomentum.
Single-cell sequencing using 10X Genomics: Single cell samples were prepared using Single Cell 3/Library & Gel Bead Kit v2 and Chip Kit (10X Genomics) according to the manufacturer's protocol. Briefly samples were FACS sorted using DAPI to select live cells followed by resuspension in 0.04% BSA-PBS. Nearly 1, 200 cells/μl were added to each well of the chip with a target cell recovery estimate of 8,000 cells. Thereafter Gel bead-in Emulsions (GEMs) were generated using GemCode Single-Cell Instrument. GEMs were reverse transcribed, droplets were broken and single stranded CDNA was isolated. cDNAs were cleaned up with DynaBeads and amplified. Finally, cDNAs were ligated with adapters, post-ligation products were amplified, cleaned up with SPRIselect. Purified libraries were submitted to UCLA Technology Center for Genomics & Bioinformatics for quality check and sequencing. The quality and concentration of the purified libraries were evaluated by High Sensitivity D5000 DNA chip (Agilent) and sequencing was performed on NextSeq500.
10× sequencing data analysis: Raw sequencing reads were processed using Partek Flow Analysis Software. Briefly, raw reads were checked for their quality, trimmed and reads with an average base quality score per position>30 were considered for alignment. Trimmed reads were aligned to the human genome version hg38-Gencode Genesrelease 30 using STAR −2.6.1 d with default parameters. Reads with alignment percentage>75% were de-duplicated based on their unique molecular identifiers (UMIs). Reads mapping to the same chromosomal location with duplicate UMIs were removed. Thereafter ‘Knee’ plot was constructed using the cumulative fraction of reads/UMIs for all barcodes. Barcodes below the cut-off defined by the location of the knee were assigned as true cell barcodes and quantified. Further noise filtration was done by removing cells having>3% mitochondrial counts and total read counts >24,000. Genes not expressed in any cell were also removed as an additional clean-up step. Cleaned up reads were normalized using counts per million (CPM) method followed by log transformation generating count atrices for each sample. Samples were batch corrected on the basis of expressed genes and mitochondrial reads percent. Count matrices were used to visualize and explore the samples in further details by generating tSNE plots generated using default parameters in Partek. K-means clustering was computed for identifying groups of cells with similar expression profile using Euclidean distance metric based on the most appropriate cluster count. A maximum of 1000 iterations were allowed and the top marker features for each cluster was determined.
Gene ontology enrichment analysis for the differentially expressed genes was performed using DAVID Gene Functional Classification Tool ([http://]david.abcc.ncifcrf.gov; version 6.8). Dot plots and Violin plots were generated in R (v4.0.3) using ggplot2 (v3.3.3) package. Two-way Venn diagrams were generated using BioVenn. Hypergeometric p values were calculated assuming 25,000 human genes.
Trajectory analysis: Cell trajectories were constructed using Monocle3 package in R according to Trapnell lab guidelines (https://] [github. com/cole-trapnell-lab/monocle3). Count matrices, cell metadata and gene metadata were used to create Monocle3 object. A subset of genes that exhibit high cell-to-cell variation in the dataset was determined by directly modeling the mean-variance relationship inherent in the data using Seurat. Preprocessing was done by calculating significant PCs (principal components) ensuring usage of enough PCs to capture most of the variation in gene expression across all the cells in the data set. Variable genes obtained from Seurat were used to pre-process the data. The dimensionality of the data was thereafter reduced using uniform manifold approximation and projection (UMAP). Cells were plotted onto the UMAP space for visualization of their distribution, identification of cell types and community detection to group cells into clusters. Next the principal graph was learned within each cluster, trajectory was constructed and the cells were ordered to measure their progress in pseudotime.
Methylcellulose culture method of porcine BMSCs and chondrocytes. To assess the clonality and capacity of pig BMSCs to undergo chondrogenesis in vitro, porcine bone marrow stromal cells (pBMSCs) or articular chondrocytes were isolated from the distal femoral epiphysis or articular surface of the condyles, respectively, of 3-4-month-old Yucatan minipigs (S & S Farms). Tissues were digested as described above. Methylcellulose-based media (StemCell) was resuspended with DMEM/F12 (Corning)+10% FBS+1% P/S/A and either 10 ng/ml FGF-2, 10 ng/ml BMP-2, 10 ng/ml TGFβ-1, or all three growth factors, to make a 1% methylcellulose-based media. Either P1 BMSCs or PO chondrocytes were seeded in 6-well ultra-low attachment plates (Corning) at a low density (300 cells/ml) and cultured for 3-4 weeks. 120-160 μL of liquid DMEM/F12 with respective growth factors was applied to the surface of the wells to ensure moisture and nutrients remained available to the cells biweekly. For the Transwell-methylcellulose co-culture, hES-derived chondrocytes on membranes were placed in a 1 μm-pore Transwell insert (Falcon) with DMEM/F12+10% FBS+1% P/S/A. P1 MSCs or P0 chondrocytes were seeded in the same media with methylcellulose, and cultured in 24-well ultra-low attachment plates (Corning) at a low density (300 cells/ml) for 3-4 weeks. 40-60 μL of media was added biweekly to the methylcellulose as maintenance. Clonogenicity was calculated by manual counting of clones larger than ˜40 μm in diameter or greater than 5 cell divisions. All images of clones in methylcellulose were taken on an Echo Revolve Inverted microscope.
ELISA detection of proteins secreted by hESDC-M. Either 1×106 human bone marrow stromal cells (hBMSCs; n=7 different donors) or whole hES-derived chondrocytes on membranes (n=6-9 batches) were lysed with 500 μL of 2× Lysis buffer (Ray Biotech) supplemented with a phosphatase/protease inhibitor (Thermo-Fisher). Lysates were then centrifuged to remove cellular debris, and a Bicinchoninic acid (BCA) protein assay (Thermo-Fisher) was performed to quantify total lysed protein. ELISAs for FGF-2 (Ray Biotech), BMP-2 (R & D Systems), and TGF-β1 (R & D Systems) were performed according to the manufacturer's protocols.
Antibody List: Please see Table 1 for a list of antibodies and dilutions used.
Detailed protocol for directed differentiation of pluripotent stem cells into chondrocytes in fully defined animal free conditions.
2D Culture: Propagation ESI-017 for MCB generation:
3D Culture: Prior to differentiation cells from the ESI-017 PSC MCB is expanded in 2D system to 1 billion cells as described above, and then transferred (as described above) into 3-D conditions/spin culture using Corning® 500 mL disposable spin flasks (Corning-3153).
Collagen Membrane with cells are cryopreserved in MesenCult ACF Freezing media (Stemcell Tech—05490) containing 10 uM Y27632 (Tocris 1254). Cells are frozen down with a controlled rate freezer, then are stored in liquid nitrogen vapor phase after 24 h in −80° C. freezer.
Reagent List and Vendor information
Cell numbers at different stages of cell differentiation
Proposed quality control testing of the intermediate/chondroprogenitors and Product/chondrocytes
Specifically, the absence of detectable levels of residual pluripotent and tumorigenic cells in the final product is determined by cell culturing and pluripotent gene expression quantitative PCR assays for the pluripotency genes Pou.5 and Lin28b. The baseline level of these genes should not exceed the levels of expression determined in fully specified human fetal chondrocytes at 8-9 weeks of development when no pluripotent cells are present in the joints.
Further, a quantitative PCR assay panel of cartilage genes is used to assess the chondrogenic activity of the product. The levels of COL2A1 gene 10-fold or higher and the levels of SOX9 gene 5-fold higher than the levels in undifferentiated ESI-017 is considered as acceptable release criteria.
Viability of the final product is assessed using quantitative CellToxTM Green (Promega CAT# G8741). Assessment of the viability is performed without digestion of the final product into single cells. Digestion of Plurocart cell sheet may lead to lower viability due in large part to the digestion step itself. The number of live cells is then calculated after measuring the amount of total and dead cells in a sample using a fluorescent plate reader. In parallel genomic DNA assay is carried out to determine the total number of cells in the tested membrane. At least 500,000 of viable cells per cm2 is used as acceptable release criteria. Stability is assessed by performing viability testing of cryopreserved product every 3 month. Viability of the product should not decline below 500,000 of viable cells per cm2 at any time of testing.
Scaled production and formulation optimization of hES-derived chondrocytes. Studies were performed to assess the long-term therapeutic potential of hESC-derived chondrocytes in a porcine model of focal articular cartilage injury. New and large-scale production methods were developed to produce articular cartilage-like chondrocytes from human PSCs. Cells generated using this technique are immature based on their transcriptional signature and expression of immature chondrocyte markers but can mature upon implantation in vivo, evidencing appropriate expression of superficial zone markers and lack of hypertrophy. For this study, procedures initially developed for H1 and H9 lines were redesigned to utilize the research grade hESC line ESI-01726. This specific line was selected because a cGMP version of this line is fully compliant with all current FDA regulations and can be advanced into human clinical trials without any regulatory restrictions.
In order to generate sufficient numbers of clinical grade hESC-derived chondrocytes for cartilage defect repair, hESCs were first expanded in hESC-qualified Matrigel and induced into mesodermal differentiation (d1-7) followed by chondrogenic differentiation (d7-11;
During the optimization stage 2 different formulations of hESC-derived chondrocytes: chondrospheres (CS) and chondrocytes integrated onto a collagen I/III membrane previously approved for clinical use (Cartimaix; Matricel), were developed and tested. For chondrosphere production, a commercially available low attachment plate with a patterned floor designed was used to generate chondrospheres of uniform size and quality from d11 MACS-purified chondrogenic cells (
Cryopreservation media (
The ability of two doses of each formulation to support short-term repair of a focal defect in pig articular cartilage were compared (
Characterization and developmental status of hESDC-M. To better understand the heterogeneity present in hESDC-M, single cell RNA-seq (scRNA-seq) was performed on cells isolated from two different batch production runs and compared them to undifferentiated ESI-017 cells (
hESDC-M support long-term repair of articular cartilage in pigs. In order to assess the therapeutic potential of hESDC-M, a long-term clinically relevant experiment was performed in which either membranes alone or membranes with cells were implanted into pig articular cartilage defects and assessed 6 months later. Pigs in each group (n=5) had 2, 6 mm full-thickness cartilage defects created with an average of 7 mm apart in their femoral condyles within the load bearing areas and were treated with either membranes alone or hESDC-M; two animals were used as sham controls with no cartilage defects generated. Cell implants were thawed and washed in fresh X-Vivo media approximately 1 h prior to implantation, applied to the defects, and were fixed in place with fibrin glue. After 6 months, pigs were euthanized and cartilage assayed using morphological, histological and biomechanical methods. Defects from all pigs transplanted with cells uniformly evidenced substantially less degeneration in and around the injury site (
At the molecular level, cartilage in the defects treated with cells more closely resembled the surrounding tissue. In membrane only defects, much of the new tissue was inferior as evidenced by collagen 1 and collagen X staining coupled with substantially reduced proteoglycan content (
hESDC-M secrete chondroinductive factors that induce chondrogenesis from porcine BMSCs. Given that the majority of hyaline-like neocartilage was contributed by pig cells, the chondroinductive, paracrine effects of hESDC-M on pig BMSCs and chondrocytes were assessed (
It is understood that various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims.
This application claims priority under 35 U.S.C. § 119 from Provisional Application Serial No. 63/193,382, filed May 26, 2021, the disclosure of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/031191 | 5/26/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63193382 | May 2021 | US |
