A factor produced by feeder cells which inhibits embryonal carcinoma cell differentiation. Characterization and partial purification
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Abstract:
Medium conditioned by STO mouse fibroblast cells inhibited both the spontaneous differentiation of NG2 embryonal carcinoma cells and the differentiation of F9 embryonal carcinoma cells induced by retinoic acid. This effect was due to a differentiation retarding factor (DRF). Reduction in DRF activity in conditioned medium by boiling and by pronase treatment suggested the involvement of a polypeptide, which had an apparent molecular weight of 57000 on gel filtration. A 28-fold purification of DRF was achieved. DRF delayed but did not prevent the extensive differentiation observed after prolonged culture of NG2 colonies. Conditioned medium could be successfully used to replace feeder cells in NG2 stock cultures. Media conditioned by a variety of other cell types also contained differentiation retarding activity.
Insights
A novel differentiation retarding factor (DRF) was identified in conditioned fibroblast cell medium. This polypeptide inhibits embryonal carcinoma cell differentiation, offering potential for stem cell culture applications.
Area of Science:
- Cell Biology
- Developmental Biology
- Stem Cell Research
Background:
- Embryonal carcinoma (EC) cells are valuable models for studying early mammalian development and differentiation.
- Maintaining EC cells in an undifferentiated state is crucial for their use in research and potential therapeutic applications.
Purpose of the Study:
- To investigate the factors present in conditioned medium that influence the differentiation of NG2 and F9 embryonal carcinoma cells.
- To identify and characterize the active component responsible for inhibiting EC cell differentiation.
Main Methods:
- Conditioned medium from STO mouse fibroblast cells was used to culture NG2 and F9 EC cells.
- Assays were performed to assess the effect of conditioned medium on spontaneous and induced differentiation.
- Biochemical treatments (boiling, pronase) and gel filtration were employed to characterize the active factor.
- Purification of the differentiation retarding factor (DRF) was carried out.
Main Results:
- Conditioned medium significantly inhibited both spontaneous differentiation of NG2 EC cells and retinoic acid-induced differentiation of F9 EC cells.
- The inhibitory activity was attributed to a differentiation retarding factor (DRF).
- DRF was identified as a polypeptide with an approximate molecular weight of 57,000 Da, sensitive to heat and pronase.
- A 28-fold purification of DRF was achieved, and it was found to delay rather than completely prevent differentiation.
Conclusions:
- A novel polypeptide, DRF, effectively inhibits embryonal carcinoma cell differentiation.
- DRF holds potential as a valuable tool for maintaining stem cell pluripotency in culture.
- Conditioned medium containing DRF can serve as a feeder-free alternative for culturing NG2 cells.