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Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF)
S Pease1, P Braghetta, D Gearing
1Ludwig Institute for Cancer Research, Melbourne Tumor Biology Branch, PO Royal Melbourne Hospital, Victoria, Australia.
Abstract:
The isolation of pluripotent murine embryonic stem (ES) cells has previously been achieved by coculturing the ES cells with fibroblast feeder cells. In this report we demonstrate that ES cell lines can be isolated from murine 129/Sv He blastocysts in the absence of feeder cells in culture medium supplemented with recombinant leukemia inhibitory factor (LIF). Three of the ES cell lines (MBL-1, MBL-2, and MBL-3) were isolated by directly explanting blastocysts, whilst two ES cell lines (MBL-4 and MBL-5) were isolated from blastocysts pretreated by immunosurgery. Three of the ES cell lines contained the Y chromosome (MBL-1, MBL-2, and MBL-5) with a high proportion of the cells displaying a normal diploid karyotype with a modal chromosome number of 40. All of the ES cell lines tested expressed the stem cell markers ECMA-7 and alkaline phosphatase, which were lost on removal of LIF when the ES cells differentiated into a variety of cell types. The full developmental potential of the ES cells was determined by injecting cells from two of the independently derived ES cell lines, MBL-1 and MBL-5, into C57BL/6J blastocysts. A high proportion of the pups born were chimeric as judged by coat pigmentation. Subsequent breeding established that the ES cells had contributed to the germ line. These results demonstrate that feeder cells are not essential for the isolation of pluripotent ES cell lines.
Insights
Pluripotent embryonic stem (ES) cells can now be isolated without feeder cells, using recombinant leukemia inhibitory factor (LIF). This breakthrough simplifies ES cell isolation and maintains their developmental potential for research.
Area of Science:
- Stem Cell Biology
- Developmental Biology
- Genetics
Background:
- Embryonic stem (ES) cells are crucial for developmental biology research.
- Traditionally, isolating pluripotent murine ES cells required co-culturing with fibroblast feeder cells.
Purpose of the Study:
- To investigate the isolation of murine ES cell lines without feeder cells.
- To determine if recombinant leukemia inhibitory factor (LIF) can support ES cell isolation and pluripotency.
Main Methods:
- Murine 129/Sv He blastocysts were cultured in medium supplemented with recombinant LIF, without feeder cells.
- ES cell lines were isolated via direct explantation or immunosurgery.
- Stem cell markers (ECMA-7, alkaline phosphatase) and karyotype were analyzed.
- Developmental potential was assessed by injecting ES cells into C57BL/6J blastocysts and evaluating chimerism and germline contribution.
Main Results:
- Five ES cell lines (MBL-1 to MBL-5) were successfully isolated using LIF without feeder cells.
- Three cell lines contained the Y chromosome and exhibited a normal diploid karyotype.
- All isolated ES cell lines expressed key stem cell markers.
- In vivo studies confirmed the pluripotency and germline contribution of the derived ES cells.
Conclusions:
- Feeder cells are not essential for the isolation of pluripotent murine ES cell lines.
- Recombinant LIF is sufficient to maintain ES cell pluripotency during isolation.
- This method simplifies ES cell derivation and expands their utility in regenerative medicine and developmental studies.