Reprograming Research Of Somatic Cells And Tumor Cells AND Research On The Application Of CRISPR-Cas9 Technology In Acute Promyelocytic Leukemia

Purpose and Background:The therapy of malignant hematological diseases is a great challenge all over the world. Although chemical therapy and radiotherapy can kill malignant cells, leukemia stem cells could escape or endure these affects, then continue proliferation and differentiation resulting in relapse. The existence of cancer stem cells (CSCs) in malignant diseases of hematology were first reported by Lapidot in 1994. Hitherto there is no unified criteria to sort and identify CSCs, as well no way to culture them in vitro over a long periodicity. In 2006, Yamanaka as well as his colleagues succeeded in inducing somatic cell into pluripotentstem cell, which is a groundbreaking discovery in the stem cell felid. This discovery open a new era of reprogramming all kinds of somatic cells (include cancer cell) through special method, which also makes the imagination about establishment of malignant disease model in vitro to study disease pathogenesis and to screen drug have the possibility to achieve. We attempt to reprogram NB4 cell in this experiment, which is an established cell line of acute promyelocytic leukemia (APL). In this process, we grasp the technology about how to reprogram. At the same time we intend to acquire induced pluripotent cancer stem cells (iPCS), make disease model and provide a platform for research of pathogenesis in APL and drug mechanism, laying the foundation extirpation of leukemia in future.Materials and Methods:1) Plat-E cells, which package murine retrovirus, were transfected with pMXs Plasmid vectors carrying different murine cDNA (Oct、Sox2、 Klf4、c-Myc) using effective transfection reagent. Then collected retrovirus and determined virus titer and infectivity. After infecting OG2-MEF cells, evaluated the efficiency of infection through Fluorescence microscope detecting GFP expression. Cultured infected OG2-MEF cells with iMEF cells. Tracing the appearance of GFP+ clone through Fluorescence microscope, then picked the well-grown colonies to establish mouse iPS cell lines and appraised their pluripotency:①Detecting colonies’ fluorescence and shape;②Alkaline phosphatase(AP)staining;③Karyotype analysis; ④Teratoma formation;⑤Immunocytochemistry;⑥Reverse Transcription Polymerase Chain Reaction(rt-PCR) detecting endogenous pluripotency gene; 2) 293T cells, which usually package human retrovirus, were transfected with pMXs plasmid vectors carrying human cDNA (Oct4、Sox2、Klf4、c-Myc) and packaging plasmid(Gag-pol and VSVG). Then collected virus, infected human foreskin (HFS) cells and evaluated the efficiency of infection by flow cytometry. Infected HFS cells were cultured with HFF-feeder cells until the colonies appeared, then picked the well-growth to establish hiPSCs cell line and appraised them as mentioned above.3) After collection and filteration of retrovirus, NB4 cells were infected in suspension every 24 hours for three times. After the last infection, the efficiency of infection was evaluated. Co-culture infected NB4 cells and HFF-feeder, the status of these cells was watched and appraised their pluripotency at morphological AP staining and molecular biology levels.Results:1) OG2-MEF cells were transformed to mES-like cells after infection and cultivation, which were flat and cloning growth, as well as had high density of granulated cells and high nuclear-cytoplasmic ratio. The pluripotency of miPSCs were identified as follows:①GFP+positive clone, resembled mESCs;②AP staining positive;③Karyotype analysis:Most analysis cells had normal karyotype respectively including 40 chromosomes;④Teratoma formation:Acquired teratomas 6-8 weeks after subcutaneously injecting miPSCs into nude mice. Then the teratomas were made into paraffin section and stained with HE. Histological examination displayed that the teratomas contained various tissues including glands (endoderm), striated muscle and fat tissue (mesoderm) and neural tissues (ectoderm);⑤Immunofluorescence:miPS cells expressed mESCs-specific surface antigens Nanog and Oct4;⑥rt-PCR:miPSCs expressed endogenous Nanog,Oct4 and Sox2 like mES cells, whereas OG2-MEF didn’t express them; 2) HFS cells were also successfully transformed to hES-resembled cells, which had sharp edge, flat and cloning growth, had high density of granulated cells and high nuclear-cytoplasmic ratio. Identification the characteristic of hiPSCs:①AP staining positive;②Karyotype analysis:Most of forty analysis cells had normal karyotype respectively including 46 chromosomes;③Teratoma formation:Acquired teratomas 8 weeks after subcutaneously injecting hiPSCs into NOD/SCID mice. Made the teratomas into paraffin section and stained with HE. Histological examination suggested that the tumor contained tissues including glands (endoderm), striated muscle (mesoderm) and epidermis (ectoderm);④ rt-PCR:hiPSCs expressed endogenous Nanog,Oct4 and Sox2 just as hES cells, whereas HFS cell didn’t; 3) With three rounds infection, the efficiency of infection is about 50%. After co-culturing infected NB4 cells with HFF-feeder, some granulated colonies appeared which were not similar to hES cells in morphology. RT-PCR detecting reprogrammed NB4 cells found only endogenous Nanog expression level was between common NB4 cells and hES cells. AP staining appeared mild positive.Conclusions:1) OG2-MEF cells could be reprogramed by Yamanaka factors. After identification of morphology, GFP fluorescence, AP staining, rt-PCR, IF, karyotype analysis and teratoma formation, the transformed cells were confirmed resemble mES cells.2) HFS cells could be induced into resemble hES cells human Yamanaka factors. After identification, these cells were testified being totally reprogrammed.3) Through three rounds infection and co-culture infected NB4 cells and HFF-feeder, NB4 cells could be partly reprogrammed via morphological, AP staining and molecular biology assay.Purpose and Background:Hematological malignant and genetic diseases is always associated with genetic changes. Gene therapy, which is accomplished through targeting modifications of mutant gene in hematopoietic stem cells, may be the key to cure leukemia. The researchers had reported the application of Homologous recombination (HR), Zinc finger endonuclease (ZFN) and transcription activator-like effector nuclease (TALEN) to cue hematological genetic diseases by genetic manipulation in animal models or cultured cells. However, there are no reports about the application of these technology in hematologic malignancies caused by acquired mutation until now. In 2013, one group reported a new engineered endonuclease (EEN) named CRISPR/Cas9, which is assembled by Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated (Cas) 9. The CRISPR-Cas9 system is an efficient, RNA-guided, site-specific DNA cleavage tool, which was successfully used for genome modifications in human, mouse, rat and Zebrafish. Here we intend to use the targeted modifications of CRISPR-Cas9 system to disturb the PML gene sequence, so that we can impede the formation of PML-RARa fusion gene, which blocks transcription and differentiation of granulocytes leading to APL, then cue the disease.Materials and Methods:1) Construction the plasmids of PX330-mcherry-PML: Designed and annealed PML-associated oligonucleotide can be cloned into the BbsI digested plasmid (PX330-mcherry) containing the entire guide RNA scaffold by T4 ligase.2) 293T cells were transfected by plasmids using Lipo2000, then the efficiency was appraised and sorted by flow cytometry (FACS). The RFP+cells were sorted and cultured for 2-4days, from which the genome was extracted to execute PCR and sent sequencing.3) NB4 cells were transfected by identified plasmids as mentioned above with nucleofection instrument, then the efficiency of transfection was appraised and sorted by flow cytometry (FACS). The RFP+ cells were sorted and cultured for 2-4days, from which genome was extracted and sent sequencing. At last the cell mass were stained by Wright’s Giemsa and analyzed morphology by microscope.Results:1)We succeeded constructing four plasmids PX330-mcherry-PML1、3、 5、6, which all have proper sequencing and be extracted.2) 293T cells’ efficiency of transfection is about 80%. Gene sequence analysis suggested that three plamids PX330-mcherry-PML-1,3,5 get targeted modifications.3) Nucleofection efficiency of NB4 cells is about 30%. Many sorted RFP+ cells were dead after cultured 2-4 days. Gene sequence analysis didn’t show any signal through three times’ repeat. The morphological changes during cultured and Wright’s Giemsa staining suggested the RFP+ cells appear the phenomenon of differentiation and apoptosis.Conclusions:We succeeded constructing PX330-mcherry-PML and also successfully disturbed PML gene sequence when we transfected them into 293T cells. However, when we nucleofected plasmid into NB4 cells and cultured the sorted cells, we found most of cells were dead and sequence analysis also was no signal. Though observing differentiation and apoptosis of RFP+ NB4 cells, we still cann’t come to a conclusion that CRISPR/Cas9 system can successfully interfere PML gene and disarm the differentiation arrest of PML-RARa fusion gene.