Chondrocyte Precursors Derived From Human Embryonic Stem Cells
This invention provides a system for obtaining cells of the chondrocyte lineage by differentiating primate pluripotent stem cells. The process involves culturing the cells as a micromass or other aggregate form in a cocktail of differentiation agents that facilitates outgrowth of the desired cell type. Progeny are capable of synthesizing Type II collagen or aggrecan, or other products that are characteristic of the chondrocyte lineage. Chondrocytes and chondrocyte precursor cells obtained according to this disclosure are suitable for use in both research and clinical therapy.
1 . A cell population obtained by differentiating primate pluripotent stem (pPS) cells, in which at least 5% of the cells synthesize either Type II collagen or aggrecan from an endogenous gene:
2 . The cell population of claim 1 , in which less than 1% of the cells synthesize elastin, Type I collagen, Type X collagen, or osteocalcin.
3 . The cell population of claim 1 , which causes closure of a 6 mm hole within 2 months in the animal model described by Koppel et al., Biomaterials 22:1407, 2001.
4 . A population of chondrocyte progenitors that proliferates in an in vitro culture, obtained by differentiating primate pluripotent stem (pPS) cells, and capable of forming progeny having the characteristics of the cells of claim 1 .
5 . The cell population of claim 1 , comprising less than 1% undifferentiated pPS cells.
6 . The cell population of claim 1 , comprising cells that have been genetically altered to express telomerase reverse transcriptase (TERT) at an elevated level.
7 . Two cell populations, comprising the differentiated cell population of claim 1 , and the undifferentiated pPS cell line from which it was obtained.
8 . A method for obtaining the cell population of claim 1 , comprising differentiating the pPS cells, then culturing the differentiated cells with a mixture of chondrocyte differentiation factors.
9 . The method of claim 8 , comprising differentiating pPS cells to form embryoid bodies.
10 . The method of claim 8 , wherein the mixture of chondrocyte differentiation factors comprises one or more factors selected from transforming growth factors (TGF), fibroblast growth factors (FGF), growth and differentiation factors (GDF), bone morphogenic proteins (BMP), hedgehog proteins (HH), L-ascorbic acid, and parathyroid hormone-related protein (PTHrP).
11 . A method for producing cartilage, comprising incubating the cell population of claim 1 under conditions where the cell population synthesizes either Type II collagen or aggrecan.
12 . A pharmaceutical composition for producing, repairing, or maintaining cartilage in vivo, comprising the cell population of claim 1 in a physiologically compatible excipient.
13 . The pharmaceutical composition of claim 12 , further comprising a matrix that is reabsorbed in vivo.
14 . A method of screening a compound for its ability to modulate chondrocyte growth, differentiation, or synthesis of cartilage components, comprising combining the compound with the cell population of claim 1 , determining any phenotypic or metabolic changes in the cell population that result from being combined with the compound, and correlating the change with an ability of the compound to modulate chondrocyte growth, differentiation, or synthesis of cartilage components.
15 . A method of reconstructing cartilage in a subject, comprising administering to the subject the cell population of claim 1 .