Study Of Somatic Cell Reprogramming Based On Cell Electrofusion

Backgroud: Reprogramming somatic cells into pluripotent cells opens up newpossibilities for transplantation therapy, the study of disease, and drug screening, throughsomatic cell nuclear transfer, transduction of defined transcription factors or somatic cellfusion with pluripotent cell. However, the problem of low efficiency, presenility and ethicalbarriers limit the application of SCNT in transplantation. And the vector of definedtranscription factors have the risk of changing genetic information, even causing cance.Cell fusion is a newly emerging method of cell engineering: two or more cells become asingle or multi-nuclear heterozygote through mediation or cell culture.2005, Cowan et al.fused human fibroblasts with human embroynic stem cells using PEG (a chemical agent), andthe features of the heterozygotes are similar to embroynic stem cells in morphology, growthcharacteristic and differentiation potency in vitro and in vivo.2009, Voldmann et al. generatedinduced pluripotent stem cells by microfluid technology. Through electrofusing mouseembrynic stem cells with mouse embroynic fibroblasts, they opened up a new perspect ofelectrofusion on iPSC. A recent report about cell fusion focused on epidemic mechanism, inwhich cell-cell fusion-mediated reprogramming may be faster (1day) and more efficient(70%) than the other methods. So cell electrofusion is a available way to somatic cellreprogramming in which cell fusion can aviod ethical problem and immunological rejectionissue like embroynic stem cells, and on the other hand, it is a feasible alternative to iPS cellgeneration compares with other methods.Objection: To electrofuse mouse somatic cells with embryonic stem cells using thecell electrofusion chip based on microfluid technology and low voltage electrofusiontechnology which designed by Bioengineering College of Chongqing University, and toobserve the biologyical feature, differential capability and DNA demethylation state offused cells. To make sure whether the fused cell is suffered reprogramming to be induced pluripotent stem cell or not. Futher more, to observe its fate under the microenviorment ofretina degeneration.Methods&Results:Part One. High throughput cell electrofusion experimentation1. Establishment cell electrofusion platform for comb-like microelectrode array chip,and observe the influence of voltage on cell activity through pulse gradient experiment. Itwas found that cells would die of irreversible perforation of cell membrance when voltage≥10V. The voltage of8V-9V is the optimal voltage for cell membrance perforation with highcell activity.2. Carring out experiments of cell aligment and electrofusion with HEK293cell,NIH3T3cell and mESCs for efficiency. The aligment efficiency of same cell was about41%, of which the different cell was about35%; The electrofusion efficiency of same cellwas about46%, of which the different cell was about59%.3. Karyotype analysis of single cell found that the chromosome number of NIH3T3cell, HEK293cell, mESC was68,72,40respectively. For confirm cell fusion, karyotypeanalysis of fused cell found that the chromosome number of NIH3T3cell&HEK293cell,mESC&NIH3T3cell was140,108respectively, which is the general number of parentcell.Part Two. Research of mouse somatic cell fused with embroynic cell andidentification its multipotentiality.1. NIH3T3cell was sorted by flow cytometer (FACS) after transfected withpZLENU-tagRFP lentivirus for red fluorescence, and then fused with embroynic stem cellexpressed GFP. The double fluorescence cell was sorted, collected and kept in stem cellculture system for stem cell-like clone.2. Fluorescence immunizing histochemistry showed that fused cell expressedmultipotential makers Oct4,Nanog,Sox2, and could differente to embryonic bodies withdouble fluorescences in vitro. Teratoma formed after fused cells was subcutaneous injectedinto nude mouse which was bigger than that of control group abviousely, but there was nostatistical difference. Paraffin section showed that tissues from three blastodermic layers wereinvolved in teratoma: Nestin (ectoderm), Desmin(mesoderm), GATA6(endoderm).Part Three. Mechanism Research of somatic cell reprogramming 1. Stem cell–related gene was detected by quantitative PCR, and it was found thatfused cell expressed four multipotential gene Oct4,Nanog,Sox2and Lin28same likemESC. The quantity of all the gene in mESC was highest, the next is fused cells, and thelast is NIH3T3. Stem cell–related protein was detected by Western-blot method, and itshowed that fused cell like mESC expressed the four protein as above.2. Methylation-specific PCR (MSP) was used to examine the methylation state of thepromoter in Oct4, Nanog gene, which showed that the promoter was demethylated in FSCas in mESC and methylated at the same time. It meant that fused cells was acquiredmultipotential characteristic at gene leve, but it was partly demethylated.Part Four. Fate of fused cell under the microenviorment of retina degeneration1. Immunological rejection was not found after fused cells were Subretinaltransplantated into RCS rats.1week after transplantated, the fused cells were survivalaccumulatively in subretina, and scattered into inner layers of retina after2month later.Nothing was found after3month. There was no statistical difference of survival rate amongthe three group.2. Transplanted fused cells could migrate, they almostly was founed beside theinjection point1week later: subretina and in outer nuclear layer; and a few migrated intoinner nuclear layer and ganglion cell layer2month later. HE staining did not find any tumoror abnormal tissue in the transplanted retina.3. Small amount of fused cells became PKCα,GFAP,chx10,nestin positive into RCSrats1week after transplantation. PKCα positive cells was fewer than GFAP positive cellswith synapse, cells with nestin positive were intensive and round or elliptic.Summary:1. High throughput cell electrofusion was performed through cell electrofusionplatform with comb-like microelectrode array chip; Cell fusion was indentificated bykaryotype analysis.2. Fused cell from mouse embronic stem cell and somatic cell expressed multipotentialmakers and had the differentiate ability in vitro and in vivo like the stem cell.3. Fused cell expressed four multipotential gene and protein Oct4,Nanog,Sox2andLin28like mESC. Promoter of Oct4,Nanog gene in mouse somatic cell was partlydemethylated afer fusion。 4. Fused cells could survive and migrate under degenerative microenviorment of retinaand could differentiate to nerve-like cell, so transplantinon of fused cell is safe and feasible.