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Published on: January 8, 2017
Establishment of an exogenous LIF-free culture system for mouse embryonic stem cells
Shumei Feng1, Lijuan Mo, Rongrong Wu
1College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, People's Republic of China.
Abstract:
Mouse embryonic stem cells (mESCs) have played a key role in the newly emerging fields of stem cell research. The traditional derivation and culture of mESCs have been based on the use of mouse embryonic fibroblasts (MEFs) treated with exogenous leukemia inhibitory factor (LIF). However, the rapid senescence of MEFs, coupled with the high cost of LIF, has significantly hampered the widespread use of mESCs in stem cell research. Thus, we present a novel exogenous LIF-free culture system for general mESCs applications, comprising fibroblast-like cells derived from the rabbit spleen (RSFs). We demonstrated that mESCs cultured on RSFs (mESCs-RSFs) maintained all mESC features after prolonged LIF-free culture, including alkaline phosphatase, cell surface markers (SSEA-1), molecular markers (OCT-4, NANOG, TERT, REX-1), karyotype, and pluripotency. The high expression level of both LIF and WNT3A in the RSFs may account for their ability to maintain mESCs without exogenous LIF. Moreover, this exogenous LIF-free culture system was verified to be of microbiological quality through analysis with electron transmission microscopy.
Insights
This study introduces a novel LIF-free culture system for mouse embryonic stem cells (mESCs) using rabbit spleen fibroblasts (RSFs). This new method maintains mESC pluripotency and key features, overcoming limitations of traditional MEF-based cultures.
Area of Science:
- Stem Cell Biology
- Developmental Biology
Background:
- Traditional mouse embryonic stem cell (mESC) culture relies on mouse embryonic fibroblasts (MEFs) and leukemia inhibitory factor (LIF).
- MEF senescence and high LIF costs hinder widespread mESC research applications.
- A need exists for more accessible and sustainable mESC culture methods.
Purpose of the Study:
- To develop and validate a novel exogenous LIF-free culture system for general mESC applications.
- To assess the ability of rabbit spleen fibroblasts (RSFs) to support mESC maintenance without exogenous LIF.
- To verify the pluripotency and quality of mESCs cultured in the new system.
Main Methods:
- Fibroblast-like cells derived from rabbit spleen (RSFs) were used as a feeder layer for mESC culture.
- Mouse embryonic stem cells (mESCs) were cultured on RSFs in a LIF-free medium.
- mESC features, including alkaline phosphatase, SSEA-1, OCT-4, NANOG, TERT, REX-1 expression, karyotype, and pluripotency, were assessed.
- Microbiological quality was verified using electron transmission microscopy.
Main Results:
- mESCs cultured on RSFs (mESCs-RSFs) maintained all essential mESC features after prolonged LIF-free culture.
- Key pluripotency markers (alkaline phosphatase, SSEA-1, OCT-4, NANOG, TERT, REX-1) and karyotype stability were preserved.
- RSFs exhibited high expression of LIF and WNT3A, potentially explaining their ability to support mESCs without exogenous LIF.
- The RSF-based culture system demonstrated microbiological quality.
Conclusions:
- Rabbit spleen fibroblasts (RSFs) provide an effective feeder layer for LIF-free mESC culture.
- This novel system successfully maintains mESC pluripotency and key characteristics, offering a viable alternative to traditional methods.
- The RSF-based LIF-free culture system is suitable for general mESC applications and has been verified for microbiological safety.
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