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WO2011065661A2 - Procédé de différenciation de cellules souches mésenchymateuses dans des cellules cartilagineuses à l'aide de dkk-1 ou sfrp-1 - Google Patents

Procédé de différenciation de cellules souches mésenchymateuses dans des cellules cartilagineuses à l'aide de dkk-1 ou sfrp-1 Download PDF

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WO2011065661A2
WO2011065661A2 PCT/KR2010/006837 KR2010006837W WO2011065661A2 WO 2011065661 A2 WO2011065661 A2 WO 2011065661A2 KR 2010006837 W KR2010006837 W KR 2010006837W WO 2011065661 A2 WO2011065661 A2 WO 2011065661A2
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dkk
sfrp
mesenchymal stem
stem cells
chondrocytes
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WO2011065661A3 (fr
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임군일
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Industry Academic Cooperation Foundation of Dongguk University
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Industry Academic Cooperation Foundation of Dongguk University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled

Definitions

  • the present invention relates to a method for producing cartilage as a tissue engineering or cell therapy, and to a method for differentiating mesenchymal stem cells into chondrocytes.
  • Articular cartilage is the most heavily loaded tissue of human tissue and is easily exposed to damage caused by wear. Articular cartilage is a free cartilage (hyaline cartilage) consisting of chondrocytes and abundant extracellular matrix. Cartilage cells occupy less than 10% of total cartilage volume and play an important role in maintaining joint cartilage because it synthesizes and secretes extracellular matrix. Reduction of chondrocyte number and chondrocyte metabolism by aging is considered to be one of the pathogenesis of osteoarthritis, a high prevalence of senile diseases (Bobacz K et al, Ann Rheum Dis, Dec, 63 (12), pp.1618-1622 , 2004). In addition, articular cartilage is a tissue that is unable to regenerate itself after injury because there is no blood vessel, nerve and lymphatic tissue. Current medical methods have a very limited method of regenerating articular cartilage normally.
  • Histologically engineered articular cartilage of differentiated chondrocytes from stem cells is essential for chondrocytes and biodegradable scaffolds, and chondrocytes are isolated from bone marrow and cultured into chondrocytes. Induce differentiation Ideal chondrocytes should have good proliferative capacity and maintain collagen type II, a specific phenotype of chondrocytes.
  • mesenchymal stem cells are known as repair cells of various types of connective tissue, and these cells can differentiate into various types of cells of the mesenchymal system.
  • mesenchymal stem cells In order to use mesenchymal stem cells to treat articular cartilage damage, it is essential to develop an efficient and well-organized method of differentiating stem cells directly into cartilage cell lines both in vivo and in the laboratory.
  • These undifferentiated mesenchymal stem cells are limited in their use because they lack information on the long-term stability of repaired tissues and are at risk of forming heterologous tissues in vivo by their tendency to differentiate into other types of cells. (De Bari, C et al., Arthritis Rheum, 50: 142, 2004).
  • the Wnt signaling inhibitor DKK-1 or sFRP-1 is treated in the early stages of differentiating mesenchymal stem cells into chondrocytes, thereby efficiently differentiating mesenchymal stem cells into chondrocytes.
  • One embodiment of the present invention provides a method for differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture.
  • one embodiment of the present invention is to differentiate the chondrocytes prepared by the method of differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture
  • compositions for treating cartilage damage diseases are provided.
  • one embodiment of the present invention is to differentiate the chondrocytes prepared by the method of differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture It provides a method for producing an artificial joint comprising continuing to culture in a scaffold of a certain type.
  • one embodiment of the present invention is to differentiate the chondrocytes prepared by the method of differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture It provides a manufactured artificial joint comprising continuing to culture in a scaffold of a certain type.
  • chondrocytes by treating DKK-1 or sFRP-1 at the beginning of differentiation of mesenchymal stem cells capable of differentiating into various types of cells, differentiation into chondrocytes can be efficiently induced. Differentiated chondrocytes prepared in this way can be usefully used to treat osteoarthritis in a biological way through the transplantation of cells or tissues.
  • 1 is a graph showing the amount of gene expression of ⁇ -catenin following treatment with DKK-1 or sFRP-1. (a) is by DKK-1, (b) is by sFRP-1.
  • FIG. 2 is a graph showing the amount of protein expression of ⁇ -catenin following treatment with DKK-1 or sFRP-1.
  • (a) is the result of western blot
  • (b) is a graph of this.
  • Figure 3 is a photograph of the results of immunohistochemical staining to determine the amount of ⁇ -catenin following treatment with DKK-1 or sFRP-1.
  • FIG. 4 is a graph showing the amount of GAG according to the treatment of DKK-1 or sFRP-1. (a) is by DKK-1, (b) is by sFRP-1.
  • FIG. 5 is a photograph of the results of safranin-O staining. (a) is by DKK-1, (b) is by sFRP-1.
  • FIG. 6 is a graph showing changes in protein gene expression essential for cartilage differentiation following treatment with DKK-1. (a) relates to COL2A1, (b) relates to SOX-9.
  • Figure 7 (a) is a result of Western blot for COL2A1 and SOX-9
  • Figure 7 (b) is a photograph of the immunohistochemical staining results.
  • One embodiment of the present invention provides a method for differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture.
  • Mesenchymal stem cells include synoviium, umbilical cord blood or adipose tissue-derived stem cells, and the like, but are not limited thereto. It is appropriate to use bone marrow-derived mesenchymal stem cells.
  • the isolated myelogenous mesenchymal mesenchymal stem cells are preferably used in two to five passages, preferably three to four times, and most preferably three times, in that they separate only stem cells that can differentiate.
  • pellet culture, alginate beads, and alginate layer culture are mainly used as a three-dimensional culture method for differentiating mesenchymal stem cells into chondrocytes.
  • pellet culture is effective in maintaining the phenotype of chondrocytes, and can easily aggregate cells through centrifugation to induce a conjugation effect between cells, thereby providing an extracellular environment similar to the production of early cartilage tissue.
  • the cells when differentiating mesenchymal stem cells into chondrocytes, the cells are cultured in the presence of DKK-1 or sFRP-1, which is preferably treated from 2 to 8 days and 3 to 7 days from the start of the culture. .
  • DKK-1 is an inhibitor of the Wnt / ⁇ -catenin signaling pathway, which is involved in the differentiation of cartilage in mesenchymal stem cells, and binds to the auxiliary receptors, LRP5 / 6 and Kremen, to inhibit Wnt signaling pathway.
  • Secreted frizzled-related protein is an inhibitor of the Wnt / ⁇ -catenin signaling pathway involved in cartilage differentiation in mesenchymal stem cells and has a cystine-rich domain for interaction with Wnt proteins. It prevents binding to the frizzled receptor on the cell membrane or LRP5 / 6, an auxiliary receptor on the cell surface.
  • Wnt is a group of several glycoproteins bound to lipids. Wnt is involved in various phenomena such as cell fate determination, polarity, and differentiation migration and plays an important role in the development of trunk and limb axis formation. It is involved in the development and formation of the skeleton in fetal development and in the remodeling of the skeleton in adults, but it has a complex mechanism of interaction with other signaling systems, depending on time and place.
  • the general Wnt signaling system is relatively simple. When Wnt binds to a receptor called frizzled on the cell membrane, it releases the cofactor, axin, from the protein complex that breaks down ⁇ -catenin, thereby inducing cytoplasmic ⁇ -catenin levels. Is increased, ⁇ -catenin enters the cell nucleus and activates the transcription factor Lef1 / Tcf-1 to induce the transcription of several genes.
  • This general Wnt / ⁇ -catenin signaling pathway constitutes an autonomic mechanism that promotes differentiation of undifferentiated mesenchymal stem cells into osteoblasts and inhibits differentiation into chondrocytes. Overexpression of Wnt leads to increased bone formation and inhibition of chondrocyte formation. On the contrary, when ⁇ -catenin is inhibited, chondrocyte differentiation occurs instead of osteoblast differentiation. Is thought to determine whether to become osteoblasts or chondrocytes.
  • the mechanism by which bone formation is promoted in the Wnt / ⁇ -catenin signaling system is a mechanism by which ⁇ -catenin-activated Lef1 / Tcf-1 transcription factors directly induce the production of Runx2, the most important transcription factor in bone differentiation.
  • Inhibition of cartilage formation is a mechanism by which ⁇ -catenin directly binds to and inactivates SOX-9, a transcription factor that induces cartilage formation.
  • Determination of cell differentiation of mesenchymal stem cells by the Wnt / ⁇ -catenin signaling system is determined by strong expression in the early stages of development and within a short time. Two days from the start of incubation of DKK-1 or sFRP-1 It is good to process for 8 to 8 days, 3 to 7 days. For example, when three-week cultures of bone marrow-derived mesenchymal stem cells that have been passaged three times after separation to differentiate into chondrocytes are treated with DKK-1 or sFRP-1 for the first week only. If the treatment period is shorter than 2 days, inhibition of the Wnt / ⁇ -catenin signaling system does not occur efficiently. If the treatment period is longer than 8 days, cell death may occur.
  • the amount of DKK-1 or sFRP-1 is suitably 50 to 350 ng / ml, 80 to 320 ng / ml, 100 to 300 ng / ml, 150 to 300 ng / ml.
  • the inhibitory effect is insignificant. If it is more than 350ng / ml, it is unnecessary to use.
  • the culture medium may include any one or more substances selected from the group consisting of insulin, dexamethasone, ascorbate 2-phosphate, L-proline and sodium pyruvate, each amount of insulin 0.5 to 3 g / l, dexamethasone ( dexametasone) 10 -6 to 10 -8 M, ascorbate 2-phosphate 10-100 mM, L-proline 10-100 mM and sodium pyruvate 0.5-3 mM.
  • one embodiment of the present invention is to differentiate the chondrocytes prepared by the method of differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture
  • compositions for treating cartilage damage diseases are provided.
  • Chondrocytes differentiated from bone marrow origin mesenchymal stem cells produced by the method according to an embodiment of the present invention can be used as an active ingredient of a cell composition for cell replacement therapy for treating cartilage damage and the like.
  • Cartilage damage diseases that can be treated using chondrocytes produced by one embodiment of the present invention include, but are not limited to, degenerative arthritis, rheumatoid arthritis, and joint damage due to trauma.
  • the therapeutic composition comprising chondrocytes produced by one embodiment of the present invention as an active ingredient may be directly injected into the joint of a patient according to a known method, or may be transplanted together with a scaffold after three-dimensional culture. It is desirable to control the number of cells to be administered in consideration of various factors such as the disease, the severity of the disease, the route of administration, the weight, age and sex of the patient.
  • one embodiment of the present invention is to differentiate the chondrocytes prepared by the method of differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture
  • one embodiment of the present invention is to differentiate the chondrocytes prepared by the method of differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture It provides a manufactured artificial joint comprising culturing in any form of scaffold.
  • Artificial cartilage of a certain type can be obtained using a conventional method of culturing chondrocytes differentiated by one embodiment of the present invention in a scaffold of any form, and by using the artificial joint or ear or nose Artificial cartilage used for molding can be produced.
  • Bone marrow samples used to isolate mesenchymal stem cells were obtained from three patients (mean age: 50 and 37-64 years) who underwent total hip replacement due to arthritis. Mesenchymal stem cells were isolated from the bone marrow and then expanded. After two passages, cell proliferation was stopped in cryopreservation medium containing 90% fetal bovine serum (Gibco BRL, Green Island, NY) and 10% dimethylsulfoxide. In subsequent experiments, the cells were thawed and one million cells were washed at a density of 1.2 * 10 4 cells / cm 2 with 10% fetal calf serum and 1% antibiotics (100 U / ml penicillin, 0.1 mg / ml streptomycin, 0.25 mg / ml).
  • DMEM Dulbecco's modified Eagle's medium
  • F-12 medium Gibco BRL, Green Island, NY
  • amphotericin B Gibco BRL, Green Island, NY
  • the medium of the experimental group contained 100 (Example 1), 200 (Example 2) and 300 (Example 3) ng / ml of DKK-1, 100 (Example 4), 200 (Example 5) and 300 (Example 6) ng / ml of sFRP-1 were added.
  • pellets were recovered for analysis.
  • 1 ml cell suspension was placed in a 15 ml polypropylene centrifuge tube and centrifuged at 500 g for 5 minutes in a centrifuge. The tubes were then stored in an incubator at 37 ° C. with 5% carbon dioxide.
  • Example 1 Example 2
  • Example 3 Example 4
  • Example 6 DMEM / F-12 medium, ITS, dexamethasone, ascorbate 2-phosphate, L-proline, sodium pyruvate ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ Amount of DKK-1 (ng / ml) 0 100 200 300 0 0 0 amount of sFRP-1 (ng / ml) 0 0 0 0 100 200 300
  • an Alamar Blue assay was performed. This assay measures cell viability using a blue non-fluorescent dye resazurin that turns into a pink fluorescent dye resorufin in response to chemical reduction of the growth medium produced by cell growth.
  • Control was DMEM containing 10% fetal calf serum and 1% antibiotics (100 U / ml penicillin, 0.1 mg / ml streptomycin, 0.25 mg / ml amphotericin B; Gibco BRL, Green Island, NY). (Dulbecco's modified Eagle's medium) / F-12 medium (Gibco BRL, Green Island, NY) was incubated in the presence of 37 °C, 5% carbon dioxide.
  • Examples 7 to 12 contained 100 (Example 7), 200 (Example 8) and 300 (Example 9) ng / ml of recombinant DKK-1 (R & D Systems) in the medium of the control group (Comparative Example 2). , Minneapolis, MN), 100 (Example 10), 200 (Example 11) and 300 (Example 12) treated with respective media to which ng / ml of recombinant sFRP-1 (R & D Systems, Minneapolis, MN) was added It became. Three times in each experimental group was incubated, the medium was changed every three days. After 3 and 6 days of incubation, 10 ⁇ l Alamar Blue TM solution (Biotium, Hayward, CA) was added and then incubated for 4 hours in an incubator at 37 ° C.
  • Alamar Blue TM solution Biotium, Hayward, CA
  • the amount of DNA was reduced by 13% compared to the control group when treated with 300ng / ml of DKK-1 and 19% compared to the control when treated with 300ng / ml of sFRP-1. However, 100 ng / ml treatment did not change or increased even more.
  • the degree of inhibition of expression of ⁇ -catenin was examined using reverse transcriptase-polymerase chain reaction for Comparative Example 1, Examples 1 to 6 after 3 days and 6 days of culture.
  • Tri-Reagent® Sigma chemical, St. Louis, Mo.
  • Quant-iT TM RNA analysis kit Qubit fluorescence intensity measurement system
  • RT-PCR 1 ⁇ g total RNA was reverse transcribed with 0.5 ⁇ g oligo (dT) primers using a RevertAid TM H Minus first-strand synthesis system (Fermentas, Hanoveer, MD). All PCRs were performed with the LightCycler 480 system® (Roche).
  • Standard 10 ⁇ l reaction was performed with 4.5 ⁇ l (10 ng) cDNA, 0.5 ⁇ l 100 mM main strand, 0.5 ⁇ l 100 mM auxiliary strand primer and 4.5 ⁇ l LightCycler 480 SYBR Green IMaster Mix (Roche Diagnostics, Mannheim, Germany).
  • the temperature was 52 ° C., and the primer sequences were 5′-CAAGTGGGTGGTATAGAGG-3 ′ and 5′-GCGGGACAAAGGGCAAGA-3 ′.
  • Example 2 In order to determine the protein expression amount of ⁇ -catenin for Comparative Example 1, Example 2 and Example 5 cultured for 6 days, it was analyzed by Western blot.
  • the isolated protein was electrophoresed on a Bio Trace TM NT nitrocellulose transfer membrane.
  • Membranes (Pall Life Science, East Hills, NY) were washed with Tris buffer solution (TBS) containing 0.1% TBS-T (tween-20) and then 2.5% nonfat dry milk (BioRad, Hercules, CA). TBS-T containing was treated for one hour. After washing the membrane three times with TBS-T, the primary antibody in TBS-T was added and incubated for 2 hours. Thereafter, the membrane was washed three times with TBS-T, horseradish peroxidase (HRP) -bound secondary antibody in TBS-T was treated for 1 hour, and the membrane was washed repeatedly. After washing three times, protein bands were observed with a chemiluminescent western blot analysis system (Amersham, Seoul, Korea).
  • TBS Tris buffer solution
  • HRP horseradish peroxidase
  • protein bands were digitized with Multi Gauge software (Fujifilm, Tokyo, Japan) and normalized to ⁇ -actin as an internal control.
  • the primary antibody was mouse ⁇ -catenin monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1: 1000 in TBS-T / 2.5% nonfat dry milk and mouse anti ⁇ -actin antibody (Sigma). , St. Louis, Mo.).
  • HRP horseradish peroxidase
  • the amount of ⁇ -catenin protein expression was reduced by about 10% and about 17%, respectively, by treatment with 200 ng / ml of DKK-1 or sFRP-1.
  • Dakocytomation LSAB2 system HRP kit (DAKO, Hambrug, Germany) was used for immunohistochemical staining.
  • the antibody was treated for 15 minutes using a microwave oven with 10 mM citric acid buffer (pH 6.0). The slides were then washed with lx wash buffer (DAKO). It was then incubated for 5 minutes with a solution without peroxidase. Monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) against rat human ⁇ -catenin in goat serum were diluted 1:50 and reacted with the subject portions overnight at 4 ° C. After washing three times with 1 ⁇ washing buffer (DAKO), horseradish peroxidase (HRP) bound secondary antibody (DAKO) was treated for 30 minutes. After washing sufficiently, the target part was reacted with the substrate buffer and the diaminobenzidine chromogen (DAKO, 50: 1) for 1 to 30 minutes and fixed.
  • DAKO horseradish peroxidase
  • the amount of GAG was inferred through a standard curve drawn with a standard solution containing chondroitin 4-sulfate extracted from bovine organs.
  • Absorbance was measured at 656 nm using Spectra max plus 348 (Molecular Devics, Sunnyvale, Calif.). The amount was divided by the amount of DNA to measure the amount of glucosaminoglycan per cell.
  • the amount of GAG expression in the cells 6 days after the culturing was higher than that after 3 days, and the effect was the greatest when treated with 300ng / ml of DKK-1 or sFRP-1. It can be seen.
  • sFRP-1 also significantly increased COL2A1 gene expression, and the effect was greater as sFRP-1 increased.
  • Example 2 In Comparative Example 1, Example 2, and Example 5, 6 days after the culture, Western blot and immunohistochemical staining for COL2A1 and SOX-9 were performed.
  • Dakocytomation LSAB2 System HRP Kit (DAKO, Hambrug, Germany) was used for immunohistochemistry.
  • DAKO Dakocytomation LSAB2 System HRP Kit
  • DBS Pepsin Soluble®
  • SOX-9 the antibody was then treated for 15 minutes using a microwave oven with 10 mM citric acid buffer (pH 6.0).
  • the slides were then washed with lx wash buffer (DAKO). It was then incubated for 5 minutes with a solution without peroxidase.
  • DAKO horseradish peroxidase

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Abstract

La présente invention concerne un procédé d'induction d'une différenciation des cellules cartilagineuses à partir de cellules souches mésenchymateuses et plus particulièrement un procédé d'induction de la différenciation de cellules cartilagineuses, qui met en œuvre une culture de cellules souches mésenchymateuses en présence de DKK-1 ou sFRP-1 qui sert d'inhibiteur de la transduction du signal wnt et une composition de traitement contenant des cellules cartilagineuses différenciées grâce audit procédé. Le procédé de différenciation des cellules souches mésenchymateuses en cellules cartilagineuses selon la présente invention utilise le traitement par DKK-1 ou sFRP-1 durant le stade précoce de la culture cellulaire, afin d'empêcher la différenciation en autres cellules durant la différenciation cellulaire initiale des cellules souches mésenchymateuses, permettant ainsi d'obtenir des cellules cartilagineuses plus facilement.
PCT/KR2010/006837 2009-11-30 2010-10-06 Procédé de différenciation de cellules souches mésenchymateuses dans des cellules cartilagineuses à l'aide de dkk-1 ou sfrp-1 Ceased WO2011065661A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014052912A1 (fr) * 2012-09-28 2014-04-03 Scripps Health Procédés de différenciation de cellules souches en chondrocytes
US9914911B2 (en) 2012-10-29 2018-03-13 Scripps Health Methods of reprogramming chondrocytes
US9974885B2 (en) 2012-10-29 2018-05-22 Scripps Health Methods of transplanting chondrocytes
CN119082014A (zh) * 2024-11-06 2024-12-06 济南万泉生物技术有限公司 一种促进线粒体增殖的培养基及方法

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KR102197871B1 (ko) * 2019-03-28 2021-01-04 이화여자대학교 산학협력단 엽산, 엽산 유도체, 또는 엽산 저해제를 이용한 줄기세포의 연골세포로의 분화 유도 방법

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JP2007267658A (ja) 2006-03-31 2007-10-18 Cardio Corp 心筋細胞の製造方法及び心筋細胞への分化誘導促進剤
KR20090069013A (ko) * 2007-12-24 2009-06-29 동국대학교 산학협력단 골수기원 중간엽 줄기세포를 연골세포로 분화시키는 방법

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014052912A1 (fr) * 2012-09-28 2014-04-03 Scripps Health Procédés de différenciation de cellules souches en chondrocytes
US10724005B2 (en) 2012-09-28 2020-07-28 Scripps Health Methods of differentiating stem cells into chondrocytes
US11859210B2 (en) 2012-09-28 2024-01-02 Scripps Health Methods of differentiating stem cells into chondrocytes
US9914911B2 (en) 2012-10-29 2018-03-13 Scripps Health Methods of reprogramming chondrocytes
US9974885B2 (en) 2012-10-29 2018-05-22 Scripps Health Methods of transplanting chondrocytes
US10179193B2 (en) 2012-10-29 2019-01-15 Scripps Health Methods of transplanting chondrocytes
US10385318B2 (en) 2012-10-29 2019-08-20 Scripps Health Method of making a population of chondrocytes from reprogrammed chondrocytes
CN119082014A (zh) * 2024-11-06 2024-12-06 济南万泉生物技术有限公司 一种促进线粒体增殖的培养基及方法
CN119082014B (zh) * 2024-11-06 2025-01-28 济南万泉生物技术有限公司 一种促进线粒体增殖的培养基及方法

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