The Induction Of Mouse IPS Cells And The Contribution Of LIF On Reprogramming

Objective To investigate the feasibility of reprogramming mouse astrocytes into induced pluripotent stem (iPS) cells.Methods Infected astrocytes with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc. Appraised them after the clones appeared.Results The clones appeared at Day 28 after the infection, the reprogramming efficiency was 0.015%±0.0005, and were positive for alkaline phosphatase activity ,OCT4 and SSEA-1 staining.Conclusion Astrocytes could be reprogrammed into iPS cell, which demonstrated the universal applicability of reprogramming. Objective To determine the effects of microRNA on nuclear reprogramming.Methods Mouse neural stem cells were induced to pluripotent stem (iPS) cells via ectopic expression of Oct4 in combination with microRNA (miR)-294 and miR-302a.Results The iPS clones were successfully obtained using Oct4+miR-294+miR-302a. Reprogramming efficiency was 0.1%, which was 7-fold greater than Oct4 alone. Moreover, induced clones expressed pluripotent markers (AP, SSEA-1, and Oct4).Conclusion These findings demonstrated that microRNAs play an important role in reprogramming, and miR-294 and miR-302a could improve efficiency of one-factor induction systems. Objective To investigate the role of LIF in the induction of mouse pluripotent stem cells.Methods 1)Different concentrations of exogenous LIF (0, 500, 1000, 1500 and 2000 U/ml) were added to the induction medium after virus removal. The number of colonies was counted on days 6, 12, 15 and 20 for each condition.2)We divided the reprogramming process into two stages. During the first stage, we added no exogenous LIF to the induction medium after virus infection. Once visible pre-iPS colonies emerged(about 6 days after virus infection), 1000 U/ml of LIF was added.3)To further investigate whether LIF is dispensable for AP activation in this system, a goat anti-murine recombinant LIF polyclonal antibody was used to block endogenous LIF, which was produced by target cells (MEF cells).Results 1)The emergence of final-iPS colonies was dependent on the concentration of exogenous LIF, which had to be at least 1000 U/ml. No ES-like colony was detected on day 20 when the concentration was less than 1000 U/ml. Notably, the numbers of final-iPS colonies formed did not differ significantly when 1000, 1500 or 2000 U/ml of LIF was used2)The pre-iPS colonies no longer detached, and final-iPS colonies were present after 21 days. These colonies were morphologically similar to mouse ES cells, including a round shape, large nucleoli and scant cytoplasm (Fig. 2A). Real-time PCR showed that the expression levels of pluripotency-associated genes in the final-iPS cells were comparable to those in ES cells (Fig. 2D). Moreover, the final-iPS cells differentiated into the three germ layers in vivo (Fig. 2B) and in vitro (Fig. 2C). 3)The successful derivation of AP-positive colonies with OSK+LIF indicated that endogenous c-Myc stimulated by LIF contributed to AP activation.Conclusion These findings illustrate a mechanism by which LIF may integrate signaling into reprogramming.