Induction of ES cell-derived cartilage formation.
This unit describes the protocols used for cultivation of murine embryonic stem (ES) cells and their differentiation into chondrogenic cell types in vitro. ES cells cultivated as cellular aggregates, so-called embryoid bodies (EBs), differentiate spontaneously into chondrogenic cell types recapitulating cellular events of chondro- and osteogenesis. The undifferentiated ES cells differentiate into mesenchymal prechondrogenic cells in the EB outgrowths. These progenitor cells aggregate and form mesenchymal condensations. During further cultivation, these cells form cartilage nodules, show a phenotype typical for chondroblasts, and start to express marker molecules of cartilage tissue. Later, the chondrocytes become hypertrophic, and finally, marker molecules indicating bone formation can be detected in the nodules. This unit also contains protocols for characterization of the differentiated cells by immunostaining, mRNA-in situ hybridization, electron microscopy, and RT-PCR analysis.
Kramer
Jan
J
University of Lübeck, Lübeck, Germany.
Schlenke
Peter
P
Rohwedel
Jürgen
J
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology*; D004396:Coloring Agents; D003470:Culture Media / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology; D020869:Gene Expression Profiling / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods; D010011:Osteocytes / Q000166:cytology; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013194:Staining and Labeling / Q000379:methods
D015415:Biomarkers; D004396:Coloring Agents; D003470:Culture Media