DNA Demethylation Of Somatic Cells During The Electro-fusion Induced Reprogramming Using A Microfluidic Chip

Background: Differentiated somatic cells can be reprogrammed into pluripotent stemcell through somatic cell nuclear transfer(SCNF), co-culture with stem cell extract,transduction of defined transcription factors and cell fusion, which has a great prospect inthe field of regenerative medicine. However, SCNF is very difficult and its efficiency isvery low, while co-culture with stem cell extract and transduction of defined transcriptionfactors both have disadvantages of time-consuming and low efficiency. In contrast, cellfusion between somatic cells and stem cells can reprogram somatic cells quickly andefficiently. Though cell fusion inducing reprogramming has defect in clinical applicationbecause fusion cells are tetraploid, it is perfect in the basic research of the mechanism ofsomatic cells reprogramming. There are three kinds of cell fusion:the chemical one such aspolyethylene glycol (PEG) inducing cell fusion, the biological one such as virus inducingcell fusion and physical one such as cell electro-fuion. All these methods have been appliedto research somatic cell reprogramming. However, PEG will cause great damage tocells,has residual toxicity and is inefficient. Virus inducing cell fusion also has a lot ofdisadvantages, for example, the virus preparation is difficult, complex operation, the titer ofvirus inactivation is of great difference and the experiment can’t be repeated. Therefore, cellelectro-fuion become an important means to study somatic cells reprogramming due to itssimple operation, no chemical toxicity, little damage and higher efficiency.Now it’s well known that epigenetic modification of somatic cells’ pluripotent genesplays a crucial role in reprogramming, which includes chromatin remodeling, histonemodification and DNA demethylation. And the DNA demethylation is the mostimportant.Although no definite demethylation enzymes have been identified sofar,AID(Activation-induced cytidine deaminase) and Tet(ten-eleven translocation) wereproved that they played a great role in DNA demethylation due to their deamination andhydroxylation respectively. Objection: To electro-fuse mouse fibroblast cells with embryonic stem cells by theimproved cell microfluid electro-fusion chip developed by our lab,sort fused cells by flowcytometry and to study the cell biology feature of fused cell.Futhermore, to confirm thesomatic cells reprogramming of fused cell and to explore the demethylation mechanism ofsomatic cell reprogramming.Methods&Results:Part One. Cell electro-fusion and study of the cell biology feature of fused cells.1. The microfluid electro-fusion chip was improved by block the non fusion zone withinsulating materials to make the electric field focus in the fusion zone and develop a highthroughput cell electro-fuion platform. The average aligment and electro-fusion efficiencyof mESCs carrying GFP and NIH3T3carrying RFP were44.35%and59.88%respectivelythrough this platform,achieveing the efficient fusion of NIH3T3/RFP and mESCs/GFP.2. The fused cells sorted by flow cytometer(FACS) had double fluorescence and couldform embryonic stem cell-like clone when cultured in stem cell culture medium. qPCRshowed that NIH3T3can’t express pluripotent genes OCT4and Nanog, and that themRNA level of OCT4and Nanog in mESCs and fused cells is high. There was nosignificant difference in statistics between mESCs and fused cells. And the mRNA level ofsomatic genes CKAP2and LaminA/C in fused cells fell89%and95%respectivelycompared with NIH3T3.Similarly, there was no significant difference in statistics betweenmESCs and fused cells.All the results suggested after the fusion of NIH3T3/RFP andmESCs/GFP,the mRNA levels of pluripotent genes and somatic genes in fused cells weresimilar with mESCs,which confirm the somatic cells reprogramming in the fused cells.Part Two. Study of demethylation mechanism of somatic cells reprogrammingbased on microfluid chip.1. Dot blot was used to detect the change of methylation state during the cell fusion,which showed that the5mC level is very high but5hmC level is very low in NIH3T3.However, the5mC level is very low and5hmC level is very high in mESCs and fused cells.Furthermore, BSP showed that the methylation percentage of promoter of OCT4and Nanogin NIH3T3is64.38%and80%respectively while0%and4%in mESCs,16.88%and20%in fused cells.And glucMS-qPCR showed that24hours,48hours,72hours and96hoursafter fusion the methylation percentage of promoter of OCT4in fused cells is85.51%,64.06%,54%and28.04%respectively. There was statistical difference of methylation percentage among the four groups.These results suggested a gradualdemethylation in fued cells.2. qPCR showed that after cell fusion the mRNA levels of AID, Tet1and Tet2in fusedcells were up-regulated,which is2.9times,10.5times and14.4times respectively as high asNIH3T3and2.1times,2.2times and2.3times respectively as high as mESCs. There wassignificant difference in statistics among the three kinds of cells. What’s more,the mRNAlevel of Tet3in fused cells fall94%compared with NIH3T3and there was on significantdifference in statistics in Tet3between fused cells and mESCs.The mRNA levels of TDGand MBD4is similar among NIH3T3,mESCs and fuse cells and there was no significantdifference in statistics. Fluorescence immunizing histochemistry also showed that mESCsand fused cells both expressed AID, but NIH3T3didn’t.These results hinted thatTet1,Tet2,AID,TDG and MBD4might involve the demethylation of fused cells.Summary:1. Using the improved microfluid chip developed by our lab, a high throughout cellelctro-fusion platform was set up which can fuse mESCs and NIH3T3efficiently toreprogram somatic cells.2. The promoter of OCT4and Nanog genes in somatic cells would demethylate aftercell electro-fusion to achieve the crucial epigenetic modification during the somatic cellsreprogramming.3. After cell electro-fusion, AID, Tet1and Tet2would up-regulate in fused cells whichmay take part in the demethylation process.