Reprogramming Of Human Foreskin Cells And NB4Cells

Background and objective:The relapse of leukemia is always a big problem all over the world. Leukemia stem cells could escape from conventional drugs which could only kill normal leukemia cells. They will acquire the ability of proliferation in suitable conditions which makes a radical cure of leukemia impossible. Leukemia stem cells are scarce and hard to be sorted. It’s also difficult for us to culture them in vitro. Recent years have witnessed the groundbreaking discovery of iPSCs (induced pluripotent stem cells). iPSC is the sixth of the league among the establishment of human ES cell line, SCNT and the identification of adult stem cells and cancer stem cells. It holds great practical value in regenerative medicine, disease model and screening of drugs. With the method of iPSCs, we want to establish iPCs (induced pluripotent cancer cells) of acute promyelocytic leukemia (APL) NB4cells, providing a platform for research of drug mechanism. There are not many scientists at home and abroad who focus on iPCs from leukemia cells. That is just our innovation.Materials and methods:1) Transfect293FT cells which package retrovirus carrying cDNA with pMX-Oct4/Sox2/Klf4/c-Myc/GFP, pMX-Gag-pol, pMX-VSVG and certain reagents. Then collect and concentrate retrovirus. After infecting human foreskin cells, evaluating the efficiency of infection (GFP expression) through flow cytometry. Culture human foreskin cells carrying foreign Yamanaka factors with MEF cells. Pick up the well-grown colonies to establish iPS cell lines and appraise their pluripotency:①AP stain②rt-PCR③Karyotype analysis④Teratoma formation.2) Culture NB4cells. After collection and concentration of retrovirus, infect NB4cells every24hours for three times.12～24hours after the last infection, evaluating the efficiency of infection (GFP expression) of NB4cells through flow cytometry. Co-culture NB4cells and HFF-feeder, change medium every other day and watch growing status of NB4cells. Appraising the pluripotency of NB4cells at morphological and molecular biology levels.Results:1) Human foreskin cells have transformed to ES-like stem cells after infection of retrovirus carrying Yamanaka factors (Oct4, Sox2, Klf4, c-Myc):colony-grown pattern, the colonies has sharp edge but the border between ES cells is vague. Results of identification of human foreskin cells are as follows:①AP stain:positive.②rt-PCR: Similarly to hES cells, reprogrammed human foreskin cells highly express endogenous Nanog, Oct4, Sox2, Klf4and c-Myc, whereas MEFs(negative control) don’t express these genes.③Karyotype analysis:50iPSCs have undergone this analysis and the result is that48of them have normal karyotype while2of them respectively has45and48chromosomes.④Teratoma formation:we acquire teratomas8weeks after NOD/SCID mice receive iPSCs subcutaneous injection. HE stains of paraffin section show us tissues from three embryonic layers:glands, muscle, and nervous tissue.2) With three rounds of retrovirus infection of NB4cells, the efficiency of infection is60%-65%. Twelve days after co-culture of NB4cells and HFF-feeder, we found that the infected NB4cells began to grow tightly together but show different growth pattern from hES cells. We conduct rt-PCR using extraction from reprogrammed NB4cells, finding that the Nanog expression level is between common NB4cells and hES cells, the expression of endogenous Yamanaka factors is the same as NB4cells.Conclusions:1) Retrovirus vectors carrying Yamanaka factors could efficiently infect human foreskin cells and transform them to ES-like iPSCs. iPSCs from human foreskin cells share similar characteristics with hES cells in many ways such as AP stain, rt-PCR and teratoma formation and they always have normal karyotype.2) Retrovirus vectors carrying Yamanaka factors could also efficiently infect NB4cells with the efficiency of60%-65%after three rounds of infection.3) After co-culture of transgenic NB4cells and HFF-feeder, NB4cells could be partly reprogrammed via morphological and molecular biology assay.