Directed differentiation of human embryonic stem cells toward chondrocytes.
We report a chemically defined, efficient, scalable and reproducible protocol for differentiation of human embryonic stem cells (hESCs) toward chondrocytes. HESCs are directed through intermediate developmental stages using substrates of known matrix proteins and chemically defined media supplemented with exogenous growth factors. Gene expression analysis suggests that the hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic culture comprising cell aggregates. At this final stage, 74% (HUES1 cells) and up to 95-97% (HUES7 and HUES8 cells) express the chondrogenic transcription factor SOX9. The cell aggregates also express cell surface CD44 and aggrecan and deposit a sulfated glycosaminoglycan and cartilage-specific collagen II matrix, but show very low or no expression of genes and proteins associated with nontarget cell types. Our protocol should facilitate studies of chondrocyte differentiation and of cell replacement therapies for cartilage repair.
North West Embryonic Stem Cell Centre, Faculty of Life Sciences, Core Technology Facility, University of Manchester, Manchester, UK.
Rachel A
RA
Oldershaw
Melissa A
MA
Baxter
Emma T
ET
Lowe
Nicola
N
Bates
Lisa M
LM
Grady
Francesca
F
Soncin
Daniel R
DR
Brison
Timothy E
TE
Hardingham
Susan J
SJ
Kimber
D000818:Animals; D002449:Cell Aggregation; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D048430:Cell Shape; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D055758:SOX9 Transcription Factor / Q000378:metabolism
D006025:Glycosaminoglycans; D055758:SOX9 Transcription Factor; C013786:A73025