Isolation Of Parthenogenetic Embryonic Stem Cells Containing Genomes From Non-Growing And Fully Grown Oocytes And Establishment Of Model Of Muscle Damnification And Repair

It is known that oocytes can be activated without male contribution in vitro and develop to blastocysts which are used to isolate parthenogenetic embryonic stem cells. Unfortunately, differentiation capacity of the parthenogenetic embryonic stem cells was rather lower than fertilized embryos derived ES cells, which might be the result of the absence of male genome. It had been found that some maternally expressed genes were repressed and some paternally expressed genes were expressed in the non-growing oocytes. Therefore, maternal genome from non-growing oocytes can partially act as "sperm genome". In the present study, parthenogenetic blastocysts containing genome from non-growing and fully grown oocytes (named as NF-pBlastocysts) were produced by germinal vesicle transfer, and three newly established parthenogenetic embryonic stem (named as NF-pES) cell lines were derived from the resulting parthenogenetic blastocysts. To verify whether possessed the potent clinic application, we had made the mouse model of muscle damage and repair. It has been shown that NF-pES cells could differentiate into muscle in vivo and take part in the muscle repair.As novel parthenogenetic embryonic stem cells, NF-pES cells not only possess the huge application potent but also provide a new tool in vitro for study the relative action of imprinted genes and organ development. Method for establishing NF-pES cells also give a good idea to successfully isolate human parthenogenetic embryonic stem cells.1. Establishment of research platform for ES cells Isolation and characterization of embryonic stem cells (ES) depending on MMC-treated MEF.Since import MMC belongs to national controlled medicine and is difficult to be purchased and price is expensive, home-made product was selected for our research. The results showed that when the concentration of 20μg/ml MMC for 4 hour or 30μg /ml for 2 hours was used, division of cells was effectively inhibited without affecting cell vitality. After MEF was treated by 30μg/ml MCC to prepare feeder layer, seven fertilized blastcysts in vivo were planted onto that layer cells. Finally, we got five embryonic stem (ES) cell lines. All the cell lines showed alkaline phosphatase (AKP) activity, Oct-4 and SSEA-1 positive. They are able to form the teratomas, which can differentiate into the tissues from three germinal layers and one cell line has ability of differentiate into sperm. Therefore, the home-made MCC not only can effectively inhibit MEF's growth, but also support growth of ES cells with potential for germ line transmission. The price of homemade MMC is very cheap, which means the experimental cost can be largely saved. In present study, we provide evidence for the application of home-made medicine in ES cell research.2. Establishment of parthenogenetic embryonic stem cells containing genomes from non-growing and fully grown oocytes and analysis of imprinted genes in NF-pES cells.Perivitelline space in GV stage oocyte is an important limited factor to affect the efficiency of enucleation in traditional GV transfer. In order to resolve this problem, a set of "zona-free" GV transplant technology was invented. The detailed methods are as follows: zona of GV-stage was removed before enucleation. All the oocytes were placed into operation solution containing 5 or 25μg/ml CCB. Only one operation arm was used to enucleate and the enucleation rate is 83.76%. GV-stage enucleated oocytes were adhered with non-growing ones through PHA-E When parameter of pulse voltage 180V, pulse width 20μs and one repeat pulse is was used, fusion rate is 62.80%. Fused reconstructed oocytes were cultured in TYH for maturation and maturation rate is 70.89%. The "zona-free" GV-transplant technology not only don't have to select oocytes by its size of Perivitelline space, reduce the possibility of destroying oocyte by cutting needle and increase efficiency of enucleation, and but also reduce the operation difficulty and is more easier for new-beginner to grasp.It had been found that some maternally expressed genes were repressed and some paternally expressed genes were expressed in the non-growing oocytes. Therefore, non-growing oocyte genome acted partially as sperm genome. In this study, "zona-free" GV transfer was applied to remove GV of 383 oocytes, and 64 MⅡstage oocytes from non-growing oocyte genome were obtained. 56 reconstituted oocytes were obtained after transferring the spindle of the resulting MⅡstage oocytes into underneath ZP. 51 reconstructed oocytes were fused. 22 reconstituted oocytes with two polar bodies and two pronuclear were cultured in vitro and developed into 13 blastocysts. 3 NF-pES cell lines were established from 13 resulting NF-pBlastocysts. All three NF-pES cell lines were positive for ES cell markers, such as alkaline phosphatase (AKP), stage-specific embryonic antigen 1 (SSEA-1) and octamer-binding transcription factor (Oct-4). They have a normal chromosome karyotype (40) and can be maintained in an undifferentiated state for extended periods of time. When NF-pES cells were injected into severe combined immunodeficient mice, teratomas with all three embryonic germ layers were obtained. The in vitro differentiation potential of NF-pES cells was analyzed by embryonic bodies (EB) formation. The expression of germ layer markers, such as nestin (ectoderm), desmin (mesoderm), and a-fetoprotein (endoderm) demonstrated that the NF-pES cells can differentiate into all three germ layers. The contribution rate of NF-pES cells in chimeric mice were examined by FACS. The result showed that contribution rate of one NF-pES-1 cell line(named NF-pES-1 cells) in heart, spleen, kidney and marrow were 15.07%, 41.02%, 3.55% and 11.53%, respectively. Contribution rate of NF-pES-2 cell line(named NF-pES-2 cells) were 83.36%, 68.74%, 29.70% and 50.44%, respectively. In control, contribution rate of one ES cell line (derived from the fertilized blastocyst) in heart, spleen, kidney and marrow were 77.96%, 84.06%, 42.49% and 52.02%, respectively. These results suggested that developmental potential of NF-pES cells is equivalent to fertilized blastocyst-derived ES cells at least in heart, spleen, kidney and marrow, which provide more evidence for application in regenerative medicine.3. Establishment of mouse model of muscle damage and repairA mass of muscle fibre in chimeric tissue and teratomas derived from NF-pES cells suggested that NF-pES cells had potential to differentiate into muscle fibre. A decrease in the number and/or function of satellite cells may lead to degenerative disorders such as Duchenne muscular dystrophy. At present, there were effective drugs for treat these patients. Recently, a proposed therapy for these disorders is myogenic cell transfer. Once an optimal protocol is established to direct the differentiation of hES cells to appropriate muscle precursors, hES cells can become an unlimited cell source for treating degenerative muscle disorders. We have established a mouse model for testing potential of NF-pES cells to differentiate into muscle fibre. NF-pES cells were induced into precursor cells, which were injected into muscle damaged site. One month later, mice were killed and muscle damaged sites were examined by immunochemistry. The results showed that precursor cells derived from NF-pES cells had inserted into muscle and differentiate into muscle fibre. Our study demonstrated that NF-pES cells possessed the potential to differentiate into muscle fibre for treating degenerative muscle disorders.