Methods and compositions for generating chondrocyte lineage cells and/or cartilage like tissue
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A technique for chondrocytes, cartilage, applied in the field of hypertrophic chondrocytes and growth plate cartilage-like tissues
Active Publication Date: 2015-12-30
UNIV HEALTH NETWORK
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Methods to generate human cartilage from pluripotent stem cells are currently lacking, but such tissues are highly ne
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example 1
[0312] result
[0313] In vitro formation of chondrocytes including induction of primitive streak (PS) populations into embryoid bodies (stage 1), specification of paraxial mesoderm in monolayer culture (stage 2), and induction of paraxial mesoderm in high-cell-density micellar media Or generation of chondrocyte progenitor cells on collagen-coated membrane filters (Stage 3), and specialized joint and growth plate chondrocytes and cartilage tissue in micelles or filter media (Stage 4) ( figure 1 A).
[0314] The first step in the differentiation from the pluripotent stem cell (PSC) state is the formation of the PS population, which in the embryo occurs during gastrulation upon formation of the three germ layers (endoderm, mesoderm and ectoderm) period. The PS population and endoderm and mesoderm subsets can be induced by PSCs using a combination of activin A (activin, Nodal's surrogate), Wnt, and BMP signaling molecules ((Nostro, Cheng et al. 2008, Kattman, Witty et al.201...
example 2
[0333] CD73+ cells represent joint non-hypertrophic chondrocytes, whereas lack of CD73 positivity allows recognition of growth plate-like hypertrophic chondrocytes.
[0334] Chondrocyte-like and cartilage-like tissues can be produced using the methods described herein, eg, the method described in Example 1. Articular chondrocytes can be isolated and / or compartmentalized from precursor or growth plate-like chondrocytes using the CD73 cell marker. As described herein, when AC-like cells are stimulated with BMP4 they become hypertrophic and lose CD73 expression on their cell surface. One method that can be used to monitor the expression of the CD73 cell surface marker is fluorescence activated cell sorting (FACS) analysis.
example 3
[0336] Use of hESC-derived chondrocytes or cartilage for drug toxicity screening
[0337] HESC-derived chondrocytes obtained using methods described herein, such as in Example 1, can be used for prospective drug toxicology screening and drug discovery. For example, typical cartilage tissue and / or hypertrophic chondrocytes of a growth plate chondrocyte-like cell line and precursors thereof can be contacted with a test substance and assayed for one or more biological endpoints, such as cell death. For example, cell death can be determined using, for example, a viable cell dye exclusion assay, such as the Trypan Blue assay described. For example, AC-like chondrocytes exposed to a test substance (drug) can be monitored for cytotoxicity after a desired time point by counting the cells permeable to the trypan blue dye. Other assays include tetrazolium salt conversion assays. Examples of such assays include the MTT assay and the WST-1 assay. The assay can be automated for high t...
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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+, PDGFR[alpha]+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 TGF[beta] 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 PDGFR[beta]+ paraxial mesoderm population with a TGF[beta]3 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.
Description
[0001] Cross References to Related Applications [0002] This is a Patent Cooperation Treaty application claiming the benefit of 35 U.S.C. §119 based on priority to U.S. Provisional Patent Application Serial No. 61 / 809,050, filed April 5, 2013, which is incorporated herein in its entirety as refer to. technical field [0003] The present disclosure relates to methods of producing chondrocytes and cartilage, and in particular articular chondrocytes and articular cartilage-like tissues and hypertrophic chondrocytes and growth plate cartilage-like tissues, from human pluripotent stem cells. Background technique [0004] The ability to efficiently and reproducibly generate differentiated cell types from pluripotent stem cells in vitro has opened the door to the development of cell-based therapies for the treatment of a wide range of degenerative and debilitating diseases. Osteoarthritis (OA) is a candidate for this treatment because it affects at least one in ten adults (Lawren...
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