[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.