Studies Related To Stem Cells Derived From Somatic Cells Nuclear Transfer Embryos And Adult Fibroblasts

Background Human embryonic stem (ES) cells possess the potential to differentiate into all the cell types which serves as a sort of repair system for the body. The remarkable potential of stem cells makes it possible to treat patients by transplanting specialized healthy cells produced from them to repair damaged and diseased body-parts. This concept is known as"stem cell therapy". Stem cell therapy is now emerging as a potentially revolutionary new way to treat disease and injury. Stem cell therapy has potential applications in treating a wide array of diseases and ailments of the brain, kidney, bone and many other tissues. However, the transplanted cells are grown from stem cells that are not genetically compatible with one patient, their immune system will reject the cells. One approach to overcome transplant rejection of human ES (hES) cells is to derive hES cells from the patient's own cells. Diverse methods, such as somatic nuclear transfer (also called therapeutic cloning) and the generation of human iPS cells (induced pluripotent stem cells) from somatic cells, have been attempted to produce genetic compatible pluripotent stem cells. These approaches were in progress.Therapeutic cloning refers to the transfer of the nucleus of a somatic cell into an enucleated donor oocyte. In theory, the oocyte's cytoplasm would reprogram the transferred nucleus by silencing all the somatic cell genes and activating the embryonic ones. The reconstructed embryos are induced embryonic developments and ES cells would be isolated from the inner cell (ICMs) of the cloned blastocysts. It is proposed that following directed cell differentiation, these cells which carried the nuclear genome of the patient could be transplanted without immune rejection for treatment of degenerative disorders among others. However, Somati cell nuclear transfer(SCNT) is still a developing technique. Success in the production of SCNT-ES cell line is attributed to optimization of several factors including the donor cell type, reprogramming time, activation protocol and use of sequential culture system. Furthermore, use of less-invasive enucleation method is suggested to be one of key factors. Today, the success rate of obtaining cloned blastocysts from this technique remains low.An approach toward the same end of the generation of"patient-specific"ES cells was recently described, in which murine fibroblasts were reprogrammed by ectopically expressing factors known to be highly expressed in ES cells. Such iPS cells were generated from mouse fibroblasts by retroviral transduction of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. Mouse iPS cells were indistinguishable from embryonic stem (ES) cells in many respects and produce germline-competent chimeras. While the four transcription factors were used to reprogram human fibroblasts to iPS cells. Application of this approach in human cells would have enormous potential and generate patient-specific pluripotent stem cells to study and potentially ameliorate human diseases. However, reactivation of the c-Myc retrovirus increases tumorigenicity in the chimeras and progeny. Moreover, the efficiency of generating human iPS cells is low, hindering clinical application.Thus, to improved the efficiency of stem cell therapy and provided necessary theory for the further study of molecular mechenisms of somatic cell reprogram, I have studied some factors on the efficiency of somatic cell nuclear transfer and generating iPS cells from mouse somatic cells.This thesis divided into six parts as follows:Part1 Effects of Different Methods of Nuclear Transfer On the Efficiency of Somatic Cell Nuclear Transfer in MiceObjective To study the effects of different methods of nuclear transfer on the efficiency of somatic cell nuclear transfer in mice, and to establish a simple, efficient method for nuclear transfer.Methods Granulosa cell was used as a donor of mouse somatic nuclear transfer.Different enucleation methods(blind aspiration,sucrose pretreatment for enucleation, chemical induced enucleation, bisbenzimide stain method) and two methods of enucleation(intracytoplasmic nuclear injection and reverse nuclear transfer)were studied on enucleation rate of the oocyte.Results The enucleation rate(88 % ) was significantly the higher in bisbenzimide stain method than in other three groups(P<0.05). The intracytoplasmic nuclear injection had a higher rate of blastocyst development and more of total cell numbers per blastocyst than those of reverse nuclear transfer method(P<0.05).Conclusion Bisbenzimide stain method and intracytoplasmic nuclear injection are efficient procedures in mouse somatic cell nuclear transfer, these procedures have the ability to maintain early nuclear transfer embryos development.Part2 Effects of Single or Combined chemical activation methods on the Activation of Mouse Somatic Cell Nuclear Transfer EmbryosObjective To establish the optimum procedure of somatic cell nuclear transfer embryos activation in mice, and a comparison has been made of the development of mouse somatic cell nuclear transfer embryos activated by single or combined chemical activation methods.Methods mouse reconstructed embryos were constructed. The development of reconstructed embryos which have activated in different methods of activation including ethanol(Eth),calcium ionophore(A23187), strontium (SrCl2) and combined these single agent treatment with 6-dimethylaminopurine(6-DMAP), cytochalasin B(CB) were observed.Results①When NT embryos were treated with 10 mmol/L SrCl2 for 6h, the activation rate (68.9%) and the rate of blastulation (7.2%) were higher than other groups(P <0.05).②The cleavage rate and total cell numbers per blastocyst were significantly higher after treated with Eth + 6-DMAP + CB ( 69.8%;46.05±2.62), SrCl2+ CB(71.9%;45.40±2.23) or A23187+6-DMAP+CB (62%;39.75±1.15) than the control group(P <0.05).Conclusion The Eth+6-DMAP+CB and the SrCl2+CB combined chemical activation methods could better activate mouse somatic cell nuclear transfer embryos.Part3 Effect of one-step or two-step culture systems on in vitro development of mouse somatic cell nuclear transferred embryosObjective To select some culture media profitable for early mouse somatic cell nuclear transfer embryos in vitro development, a comparison has been made of the development of mouse somatic cell nuclear transfer embryos in either one-step or two-step culture systems.Methods Mouse reconstructed embryos were done. Following activation, NT embryos were allocated randomly to one of two treatment groups. Group 1, NT embryo development was compared in: KSOMgAA, KSOMgAA / KSOMgAA, cleavage medium and cleavage medium/blastocyst medium. Second group, NT embryos were cultured in medium KSOMgAA and KSOMgAA +Taurine. The development of NT embryos in one-step and two-step culture systems was observed, respectively.Results There were no significantly differences in the proportion of blastocysts, hatching rate, and total cell numbers between one-step and two-step culture systems. Development of early reconstructed and parthenogenetic embryos was significantly increased by adding taurine in KSOMgAA culture medium when compared to KSOMgAA media.Conclusion Both one-step and two-step culture systems were feasible mouse somatic cell nuclear transffer embryos culture method in vitro. Part4 Microsatellite DNA analysis of mouse somatic cell nuclear transfer blastocystsObjective To identify the source of the somatic cell nuclear transfer blastocysts, microsatellite DNA assay was developed.Methods DNA isolation from SCNT blastocyst,donor BALB/c mouse, recipient C57BL/6 mouse and KM mouse were tested by nested polymerase-chainreaction(PCR) analysis. Primers designed for four specific microsatellite locus (D3Mit28, D11Mit258, D12Mit136, D14Mit50) were employed.Results Microsatellite DNA analyses examining four loci confirm that all the SCNT blastocysts were genetically identical to the donor mouse. Additionally, the SCNT embryos were not genetically related to the respective recipient mouse. Furthermore sequences of SCNT blastocysts are diferent from the control KM mouse.Conclusion The results have proved that the nucleus of somatic cell nuclear transfer blastocysts come from somatic nucleus of donor BALB/c mouse.Part5 Isolation of Embryonic Stem Cell-like Colonies From Mouse Nuclear Transfer EmbryosObjective The aim of the experiment was to isolate and culture mouse ES cell-like colonies from mouse nuclear transfer embryos, and to compare some factors influencing the efficiency of establishing those stem cell-like colonies.Methods Mouse NT embryos were done. When NT embryos developed to the stage of early blastocyst,embryos were transferred to the feeder layer of mouse embryo fibroblast with cardiomyocyte media conditioned for 4-5 days,and then ICMs were digested and recultured. ES cell-like colonies were isolated after 2 days,and the morphology of this colonies were observed.Results Outcome of isolating ES cell-like colonies was good. And the ES cell-like colonies had its typical morphological characteristics such as nest-like colonies,round or elliptic,with clear edge and smooth surface,strong refraction,significant swelling growth, compactness of cell aggregation,and obscure distinction between two edges of neighbour cells within a colony,relatively bigger nucleus compared with little cytoplasm.ES cells showed strong AKP activity. The differentiation of ES cells is outgrowth from embryonic bodies in vitro, while glandular tissues, scalelike cylindrical epithelium and muscular tissue were formed from ES cells in vivo.Conclusions This result demonstrates that ES cell-like colonies can be isolated from mouse NT embryos.Part6 Induction of Pluripotent Stem Cells from Mouse Adult FibroblastObjective This present study aimed to reprogramm mouse fibroblasts into a pluripotent ES-cell-like state in vitro.Methods The NIH-3T3 cells were infected with Pou5f1-, Sox2-, c-Myc- and Klf4-expressing retroviral vectors respectly. The mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) were seeded on feeders and incubated in the virus/polybrene-containing supernatants. Three days after infection, we added G418. Clones were selected for 2 to 3 weeks. Results Most colonies had a flat morphology and 4 colonies were ES-like when selection was applied early, the number of ES-like colonies (11) that increased at later time points. ES-like colonies isolated were with island-like images.Conclusions This demonstrates that the activity of Pou5f1, Sox2, Klf4 and c-Myc can apparently turn mouse somatic cells into cells that characteristic morphology closely resemble ES cell colony.