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Updated: Jul 1, 2026
Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells
Published on: June 21, 2012
Human feeder cells support establishment and definitive endoderm differentiation of human embryonic stem cells
Jing Zhou1, Qi Ou-Yang, Jin Li
1Institute of Reproductive and Stem Cell Engineering, Central South University and National Engineering and Research Center of Human Stem Cells, Changsha, China.
Abstract:
Mouse embryonic fibroblasts (MEFs) have been extensively used as feeder cells to support the in vitro propagation of human embryonic stem cells (hESCs). However, owing to the risk of cross-contamination with animal or other unknown pathogens, the use of MEFs does not meet requirements for the clinical application of hESCs. Moreover, the actual role played by the feeders in the differentiation of hESCs is still unclear. In this study, human embryonic fibroblasts (HEFs) were used as feeder cells to support the establishment and undifferentiated growth of hESCs, and the capability of HEFs to induce the differentiation of definitive endoderm (DE) was evaluated. Three new hES cell lines were derived. These cell lines exhibited and maintained the common features of traditional hESCs after prolonged culture in vitro. Furthermore, DE differentiation of the newly established hES cell lines was performed using 100 ng/ml activin A, and the effects were compared among HEFs, MEFs, and feeder-free systems. On day 5 of induction, DE (SOX17(+)) cells appeared with comparable efficiency in both human and mouse feeder systems (85.0 +/- 8.9% and 78.7 +/- 3.4%, respectively). These levels were considerably superior to that obtained in the feeder-free system (22.7 +/- 5.6%). The SOX17(+) cells tended to differentiate into an endodermal lineage in vivo and could be further induced into glucagon and C-peptide double positive islet-like clusters in vitro. Our studies suggest that, in terms of therapeutic application, HEFs can be an effective substitute for MEFs for sustaining the derivation and DE differentiation of hESCs.
Insights
Human embryonic fibroblasts (HEFs) effectively support human embryonic stem cell (hESC) derivation and definitive endoderm (DE) differentiation, offering a safer alternative to mouse feeders for clinical applications.
Area of Science:
- Stem Cell Biology
- Developmental Biology
- Cell Culture Technology
Background:
- Mouse embryonic fibroblasts (MEFs) are standard feeder cells for human embryonic stem cells (hESCs) but pose contamination risks for clinical use.
- The role of feeder cells in hESC differentiation remains incompletely understood.
- Clinical applications of hESCs necessitate safer, animal-component-free culture systems.
Purpose of the Study:
- To evaluate human embryonic fibroblasts (HEFs) as an alternative feeder layer for hESC culture and differentiation.
- To assess the efficiency of HEFs in supporting the derivation and undifferentiated growth of new hESC lines.
- To compare the capability of HEFs, MEFs, and feeder-free systems in inducing definitive endoderm (DE) differentiation from hESCs.
Main Methods:
- Derived three new hESC lines using HEFs as feeder cells.
- Cultured hESCs on HEFs, MEFs, and in feeder-free conditions.
- Induced definitive endoderm (DE) differentiation using activin A (100 ng/ml) and quantified SOX17(+) cells.
Main Results:
- Established hESC lines maintained typical hESC characteristics after prolonged culture.
- HEFs and MEFs supported comparable DE differentiation efficiency (85.0% and 78.7%, respectively), significantly higher than feeder-free systems (22.7%).
- Induced DE cells differentiated into endodermal lineages in vivo and formed islet-like clusters in vitro.
Conclusions:
- Human embryonic fibroblasts (HEFs) are a viable and safer substitute for mouse embryonic fibroblasts (MEFs) in hESC culture.
- HEFs effectively support both the derivation and definitive endoderm (DE) differentiation of hESCs.
- These findings support the therapeutic application of hESCs cultured with HEFs.
