Generation Of Induced Mouse Pluripotent Stem Cells Using Small Molecules In Fully Chemically Defined Medium

Currently, there are mainly three methods to reprogram somatic cells into induced pluripotent stem cells, including cell fusion, nuclear transfer and induced pluripotent stem cell techniques(iPSCs). In recent years, iPSCs technique had become a very popular approach to obtain pluripotent stem cell in vitro. This method is to apply with specific transcription factor to reprogram somatic cells into iPSCs which had similar characteristics of embryonic stem cells. However, this technique may cause the viral vector sequence and exogenous transcription factor sequence permanently integrated into the cell genome. Afterward, iPSCs seem to have normal morphology, but there may be some security risks since foreign genes could integrate with host genome and may be overexpressed. The shortage of this method can severely limit the application of iPSCs in basic and clinical researches. Therefore, plenty of studies had been commited to finding safe method for introducing exogenous gene and minimizing the use and expression of exogenous transcription factors. Previous study reported that mouse iPSCs could be generated by applied with small molecule compounds without using any viral vector and foreign transcription factors, termed chemically induced pluripotent stem cells(CiPSCs). This approach avoids the integration of foreign genes into genome and bio-security issues brought to the cells, and this complete chemical method opens up a new direction for cell reprogramming. However, for this reprogramming method, there are still two aspects to be considered, longer time consume on reprogramming and the interference of serum to the study of reprogramming mechanism. For overcoming these barriers, we will establish serum-free small molecules inducing system to generate mouse iPSCs, and optimize this inducing system. Through adjusting the composition and concentration of the small molecule compounds, we optimized the small molecule compounds inducing system. Meantime, we also analyze the biological characteristics of these mouse iPSCs we obtained. This study potentially lay the foundation for futher study of the mechanism of small molecules reprogramming. The results show as following:1. Preliminary establishment of mouse small molecule compounds induced pluripotent stem cells system. We applied with N2B27 addition with seven small molecule compounds(CHIR98014, Forskolin,616452, DZNep, VPA, Tranylcypromine, TTNPB) to induce MEF reprogramming. Their primary colonies were formed at day 8. Their morphology were similar to that of embryonic stem cells, presented a three dimensional multilayer structure, and almost these cells had high ratio of nuclear to cytoplasmic. Currently,we had established four CiPSCs-like cell lines which had been currently passaged over ten times and remained the original form of colony.2. Preliminary identification of the biological characteristic of mouse CiPSCs. The result showed that mouse CiPSCs had high activity of alkaline phosphatase(AP) and expressed OCT4, SOX2, E-cadherin and SSEA-1 determined by immunofluorescence, but expression of Nanog was not detected. The result of PCR detection showed that these cells expressed OCT4, SOX2, KLF4, CDH1, DNMT3B, DAPP4, REX1. Meantime, the maker proteins of three germ layers, SMA for Mesoderm, beta-tubulin for Ectoderm, SOX17 for Endoderm, and the marker genes of three germ layers, FOXA2, HNF4A for Endoderm, NES, TUBB3 for Ectoderm, MEF2C, BMP4, VIM for Mesoderm were detected at those spontaneously differentiated CiPSCs-like cells. It indicates that those mouse CiPSCs could spontaneously differentiate into three germ layers. Then we also determined the methylation level of Nanog promoter region. The result showed that the methylation level of Nanog promoter region decreased while the passage number of mouse CiPSCs increased.3. Optimization of the components and concentration of small molecule compounds on the generation and passage of mouse CiPSCs. We explored the effect of different concentrations of CHIR98014 on formation of primary colonies. The result showed that the concentration of 2.5uM CHIR9814 were more effective to generate primary colonies. The reprogramming efficiency was 3-5 times higher than that of the concentration of 10 or 20uM. We also found that thawed mouse CiPSCs were more efficient to form colonies by using 4i/LIF(CHIR98014, Forskolin,616452, PD0325901) medium instead of 7i. It indicates that 4i/LIF is more suitable form CiPSCs passage than 7i after thawing. It also seems that some molecules in 7i were harmfull to mouse CiPSCs at this stage.In conclusion, under our laboratory conditions, we have initially established and optimized the serum-free, chemical composition clearly and completely small molecule compounds inducing system for the generation of mouse CiPSCs, and successfully reduced the time consume for primany colony formation to 8 days. There are four mouse CiPSCs-like cell lines have been established, and their pluripotency have been partially proved.