The Establishment Of Embryonic Stem Cells And The Studies Of Hematopoietic Differentiation Of ES Cells

Part ?: the Etablishment of Mouse Embryonic Stem Cell and the Characteristics of Human Parthenogenetic Embryonic Stem CellChapter ?: the Etablishment of Mouse Embryonic Stem Cell[Objective]To establish mouse embryonic stem cells for the further hematopoietic differentiation.[Materials and methods]1. We used the mice of 129/sv(female)and OG2(male) to establish fertilized embryonic stem cells(F-ES). NT-ES cell was derived from the testicular sertoli cells from 129OG2F1 mice used as donor nuclei for SCNT. The characteristic of OG2 mouse is that the gene of Oct4 fuses with the gene of GFP.2. We use the regulatory method to establish the cell line: obtained the dpc3.5 mice, washed the uteruses and got the embryos, plated them on the feeder layer, passage after 4-6 days culture.[Result]We obtained 16 embryos from 3 mice of dpc3.5,including 9 blastocysts, 5 morulas, 2 bad embryos. At last, we got 2 cell lines from the blastocysts, and none of others. We identified one of the two cell lines and found out that the cell line consistented completely with characteristics of embryonic stem cells.[Conclusion]The ES cell line we established is consistent completely with characteristics of ES cells.ChapterП: the characteristics of human parthenogenetic embryonic stem cell[Objective]Through the identification of the characteristics of human parthenogenetic embryonic stem cell, we obtain the techniques of hES cell culture and learn some characteristics of phES cell.[Materials and methods]1. The phES cell was offered by the third affiliated hospital of Guangzhou medical college.2. The methods of passage we used: mechanical method and trypsinization.3. We tested the markers of Oct4, SSEA-2,SSEA-4, TRA-1-60, TRA-1-81 and performed the tests of EB formation, karyotype analysis, STR and teratoma formation.[Result]1. The phES cell line can express all the markers of Oct4, SSEA-2,SSEA-4, TRA-1-60, TRA-1-812. It can form EB. Karyotype is 45, X0 and the loci tested by STR is consistent completely with the donor.3. We have repeated the teratoma testing 3 times and the testing is still negative.[Conclusion]The phES cell line culture method is consistent with the hES cell line. The characteristics of phES cell line is abnormal at the karyotype and teratoma testing. PartП: Hematopoietic Differentiation of ES CellsChapter ?: Hematopoietic Differentiation of Nuclear Transfer Embryonic Stem Cells[Objective]To study the hematopoietic potential of NT-ES cells .[Materials and methods]1. The NT-ES and F-ES cell lines were offered by national institute of biological sciences,Beijing.Both of the cell lines were used to hematopoietic differentiation after passage to 18.2. The steps of hematopoietic differentiation was predifferentiation, primary differentiation and secondary differentiation. We campared the characterization of NT-ES and F-ES cell lines during the process.3. Tranplantation: to test whether the hematopoietic stem cells derived from NT-ES and F-ES cells had the hematopoietic potential in vivo.[Result]1. The total EBs of NT-ES and F-ES are 129 and 134; The total hematopoietic EBs of them are 73 and 78.2. We tested the double positive cells of CD34 and Sca-l by FACS. The rates of NT-ES are 6.58%,32.03%,23.44%,and F-ES cells are 10.35%,32.88%,16.54% respectively tested at EB-day5, EB-day7 and EB-day10。3. The result of Realtime PCR showed NT-ES and F-ES cells almost had same trend during the hematopoietic differentiation.4. The total colonies are 37 and 32 of NT-ES and F-ES in BL-CFC assay.5. Both of the NT-ES and F-ES showed positive in hematopoietic transplantation.[Conclusion]NT-ES and F-ES cell lines have almost same hematopoietic potential in vitro and in vivo. ChapterП: Initially Studying the Role of BMP4 to Hematopoietic Differentiation of Human Embryonic Stem Cell[Objective]To study the role of BMP4 to the Hematopoietic differentiation of human embryonic stem cells, and offer some useful information to the hematopoietic transplantation of human.[Materials and methods]1. The hES1 cell line was offered by the third affiliated hospital of Guangzhou medical college.2.The experiments were divided into 3groups: control(without cytokines), cytokine(adding routine cytokines ) and Bmp4(Bmp4+ routine cytokines).3.We compared the difference in EB formation by mechanical method and trypsinization.[Result]1. At EB-day12, We tested the markers of CD34,CD117 and C38 by FACS. The rates of CD34+CD38-are 1.63%,4.77%,9.83% respectively to control, cytokine and Bmp4 group;The rates of CD34+CD38+ are 1.48%,2.13%,2.80%;The rates of CD34+CD117+ are 3.09%, 4.40%, 9.91% respectively.2. The total colonies are 8, 26 and 35 respectively to control, cytokine and Bmp4 group in CFC assay. The result of Realtime PCR showed EG-GFP+ cell was a little more active than EG-GFP- cells during the hematopoietic differentiation.3. Mechanical method was better than trypsinization in EB formation.[Conclusion]1. Bmp4 can enhance the hematopoietic potential of hES cell obviously.2. Mechanical method was better than trypsinization in EB formation.