[Study on growth and differentiation of ES cells transfected with LIF gene].
We constructed plasmids pSVLD(+) and pSVLD(-) containing human D-form Leukemia Inhibitory Factor (LIF) cDNA sequence in sense or antisense orientation, transfected them into cells of an embryonic stem cell line ES-5, and isolated 248 pSVLD(+)-transfected and 93 pSVLD(-)-transfected G 418-resistant clones. By stepwise reducing LIF concentration in the medium, we obtained 3 pSVLD(+)-transfected clones (A 1-3) that could grow in 15% BRL-CM, including ESL(+)A 2 that could grow without LIF: we also obtained 13 pSVLD(-)-transfected clones (B 1-13) which would differentiate in 60% BRL-CM, including ESL(-)B 3 and B 5 that could not be passaged without LIF. ESL(+)A 2 and ESL(-)B 5 cells had the relatively stronger LIF mRNA or antisense LIF RNA expression, and LIF overexpression in ESL(+)A 2 cells was shown by biological assay for ES cell differentiation inhibition. ESL(+)A 2 cells could be continuously passaged for at least 13 passages without addition of exogenous LIF, retained undifferentiated morphology as well as a high growth rate, and resembled ES-5 cells in terms of stem cell characteristics and pluripotent properties, as analyzed for alkaline phosphatase activity and with staining the paraffin sections of tumor formed by inoculating ESL(+) A 2 cells into mouse. On the contrary, ESL(-) cells should be cultured in higher concentration of LIF than ES-5 cells, otherwise, would undertake extensive differentiation. By hanging drop culture for 3 days in the presence of 10(-6) mol/L RA then observing the differentiation of the formed embryonic bodies (EBs), we found that ESL(+) A 2 and ES-5 cells underwent similar morphologically differentiation, with round and epitheliallike cells occurring around the EBs; while ESL(-) B 5 cells, despite initial differentiation to round cells, differentiate into fibroblast-like and spindle shaped cells. The above results indicate that LIF overexpression in ESL(+) A 2 cells is able to completely free ES cells from the dependence on LIF-conditioned medium, and endogenous LIF gene expression, although is very low, may be indispensable for inhibiting the differentiation in vitro of ES cells; LIF overexpression might not obviously change the differentiation way of ES-5 cells, however, blocking endogenous LIF expression gives rise to the increased sensitivity of ES-5 cells to differentiate, with an altered differentiation pattern. The establishment of ESL(+) and ESL (-) cell lines provides models for further study of the growth and differentiation of ES-5 cells.
Shanghai Institute of Cell Biology, Academia Sinica, China.
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D000818:Animals; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D005333:Fetus / Q000166:cytology; D015870:Gene Expression; D006131:Growth Inhibitors / Q000235:genetics*; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines / Q000235:genetics*; D051379:Mice; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000096:biosynthesis; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014162:Transfection
D006131:Growth Inhibitors; D015850:Interleukin-6; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D008222:Lymphokines; D016372:RNA, Antisense; D012333:RNA, Messenger