公开(公告)号：US20230330152A1

公开(公告)日：2023-10-19

申请号：US18336410

申请日：2023-06-16

Applicant: UNIVERSITY HEALTH NETWORK

Inventor： Gordon KELLER ,  April M. CRAFT

IPC: A61K35/32 ,  C12N5/077 ,  G01N33/50

CPC classification number: A61K35/32 ,  C12N5/0655 ,  G01N33/5044 ,  C12N2501/115 ,  C12N2501/155 ,  C12N2501/16 ,  C12N2501/415 ,  C12N2501/999 ,  C12N2503/02 ,  C12N2506/02 ,  C12N2506/03

Abstract: A method for generating chondrocytes and/or cartilage, optionally articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue and/or hypertrophic chondrocyte like cells and/or cartilage like tissue, the method comprising:

a. culturing a primitive streak-like mesoderm population, optionally a CD56+, PDGFRalpha+ KDR- primitive streak-like mesoderm population, with a paraxial mesoderm specifying cocktail comprising:

i. a FGF agonist;
ii. a BMP inhibitor; optionally Noggin, LDN-193189, Dorsomorphin; and
iii. optionally one or more of a TGFbeta inhibitor, optionally SB431524; and a Wnt inhibitor, optionally DKK1, IWP2, or XAV939;

to specify a paraxial mesoderm population expressing cell surface CD73, CD105 and/or PDGFR-beta;
b. generating a chondrocyte precursor population comprising:

i. culturing the paraxial mesoderm population expressing CD73, CD105 and/or PDGFR-beta at a high cell density optionally in serum free or serum containing media;
ii. culturing the high cell density CD73+, CD105+ and/or PDGFRbeta+ paraxial mesoderm population with a TGFbeta3 agonist in serum free media to produce a high cell density Sox9+, collagen 2+ chondrocyte precursor population; and


c. either

i. culturing the high cell density Sox9+, collagen 2+ chondrocyte precursor population with the TGFbeta3 agonist for an extended period of time to produce an articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue; or
ii. culturing the high cell density Sox9+ collagen2+ chondrocyte precursor population with a BMP4 agonist for an extended period of time to produce a hypertrophic chondrocyte like cells and/or cartilage like tissue.

公开(公告)号：US20190111086A1

公开(公告)日：2019-04-18

申请号：US15977536

申请日：2018-05-11

Applicant: UNIVERSITY HEALTH NETWORK

Inventor： Gordon KELLER ,  April M. CRAFT

IPC: A61K35/32 ,  C12N5/077 ,  G01N33/50

Abstract: A method for generating chondrocytes and/or cartilage, optionally articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue and/or hypertrophic chondrocyte like cells and/or cartilage like tissue, the method comprising: a. culturing a primitive streak-like mesoderm population, optionally a CD56+, PDGFRalpha+ KDR− primitive streak-like mesoderm population, with a paraxial mesoderm specifying cocktail comprising: i. a FGF agonist; ii. a BMP inhibitor; optionally Noggin, LDN-193189, Dorsomorphin; and iii. optionally one or more of a TGFbeta inhibitor, optionally SB431524; and a Wnt inhibitor, optionally DKK1, IWP2, or XAV939; to specify a paraxial mesoderm population expressing cell surface CD73, CD105 and/or PDGFR-beta; b. generating a chondrocyte precursor population comprising: i. culturing the paraxial mesoderm population expressing CD73, CD105 and/or PDGFR-beta at a high cell density optionally in serum free or serum containing media; ii. culturing the high cell density CD73+, CD105+ and/or PDGFRbeta+ paraxial mesoderm population with a TGFbeta3 agonist in serum free media to produce a high cell density Sox9+, collagen 2+ chondrocyte precursor population; and c. either i. culturing the high cell density Sox9+, collagen 2+ chondrocyte precursor population with the TGFbeta3 agonist for an extended period of time to produce an articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue; or ii. culturing the high cell density Sox9+ collagen2+ chondrocyte precursor population with a BMP4 agonist for an extended period of time to produce a hypertrophic chondrocyte like cells and/or cartilage like tissue.

公开(公告)号：US20160038544A1

公开(公告)日：2016-02-11

申请号：US14782070

申请日：2014-04-02

Applicant: UNIVERSITY HEALTH NETWORK

Inventor： Gordon KELLER ,  April M. CRAFT

IPC: A61K35/32 ,  C12N5/077 ,  G01N33/50

CPC classification number: A61K35/32 ,  C12N5/0655 ,  C12N2501/115 ,  C12N2501/155 ,  C12N2501/16 ,  C12N2501/415 ,  C12N2501/999 ,  C12N2503/02 ,  C12N2506/02 ,  C12N2506/03 ,  G01N33/5044

Abstract: A method for generating chondrocytes and/or cartilage, optionally articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue and/or hypertrophic chondrocyte like cells and/or cartilage like tissue, the method comprising: a. culturing a primitive streak-like mesoderm population, optionally a CD56+, PDGFRalpha+ KDR-primitive streak-like mesoderm population, with a paraxial mesoderm specifying cocktail comprising: i. a FGF agonist; ii. a BMP inhibitor; optionally Noggin, LDN-193189, Dorsomorphin; and iii. optionally one or more of a TGFbeta inhibitor, optionally SB431524; and a Wnt inhibitor, optionally DKK1, IWP2, or XAV939; to specify a paraxial mesoderm population expressing cell surface CD73, CD105 and/or PDGFR-beta; b. generating a chondrocyte precursor population comprising: i. culturing the paraxial mesoderm population expressing CD73, CD105 and/or PDGFR-beta at a high cell density optionally in serum free or serum containing media; ii. culturing the high cell density CD73+, CD105+ and/or PDGFRbeta+ paraxial mesoderm population with a TGFbeta3 agonist in serum free media to produce a high cell density Sox9+, collagen 2+ chondrocyte precursor population; and c. either i. culturing the high cell density Sox9+, collagen 2+ chondrocyte precursor population with the TGFbeta3 agonist for an extended period of time to produce an articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue; or ii. culturing the high cell density Sox9+ collagen2+ chondrocyte precursor population with a BMP4 agonist for an extended period of time to produce a hypertrophic chondrocyte like cells and/or cartilage like tissue.

Abstract translation: 一种用于产生软骨细胞和/或软骨的方法，任选地关节像非增生性软骨细胞和/或软骨样组织和/或肥大软骨细胞样细胞和/或软骨样组织，所述方法包括：a。 培养原始条纹样中胚层群体，任选地是CD56 +，PDGFRalpha + KDR-原始条纹状中胚层群体，其中傍轴中胚层指定混合物，包括：i。 FGF激动剂; ii。 BMP抑制剂; 任选地，Noggin，LDN-193189，Dorsomorphin; 和iii。 任选地一种或多种TGFβ抑制剂，任选的SB431524; 和Wnt抑制剂，任选地DKK1，IWP2或XAV939; 指定表达细胞表面CD73，CD105和/或PDGFR-β的近轴中胚层群; b。 产生软骨细胞前体群体，包括：i。 以高细胞密度任选地在无血清或含血清培养基中培养表达CD73，CD105和/或PDGFR-β的近轴中胚层群; ii。 在血清游离培养基中培养具有TGFbeta3激动剂的高细胞密度CD73 +，CD105 +和/或PDGFRβ+近轴中胚层群体以产生高细胞密度Sox9 +，胶原蛋白+2软骨细胞前体种群; 和c。 我是 用TGFbeta3激动剂长时间培养高细胞密度Sox9 +，胶原蛋白+软骨细胞前体种群，以产生关节性非肥大软骨细胞和/或软骨样组织; 或ii。 用BMP4激动剂长时间培养高细胞密度的Sox9 +胶原蛋白2 +软骨细胞前体群体，以产生肥大的软骨细胞样细胞和/或软骨样组织。