Establishment And Characterization Of Human Embryonic Stem Cells Line And The Research For Comparison Of Different Cryopreservation Of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) are derived from the pluripotent cells of theinner cell mass (ICM) of the human blastocysts. These cells can be propagatedindefinitely in vitro, yet still retain a normal karyotype and the capacity fordifferentiation into a wide variety of somatic tissues representing progeny of the threeembryonic germ layers. The establishment of hESCs lines is expected to havefar-reaching applications in the field of regenerative medicine, pharmacology andbasic scientific research. Recently, many researches concerning about theestablishment of hESCs lines have been done by many different organizations. D₃embryos were usually transferred in traditional IVF-ET (in vitro fertilization andembryo transfer) procedure. After IVF-ET, many embryos of bad-quality which maybe discarded were collected and cultured in G2.3 for 2-4 days. Some of these embryoscan form blastocysts of different quality. Using the ICM of these blastocysts and withthe human embryonic pulmonary fibroblasts (HEPFs) as feeders, we have donesome research about the establishment and characterization of hESCs lines. Somepatients who had succeeded in delivering healthy children would like to donate theirsuperfluous frozen embryos. With the blastocysts cultured from these thawedembryos we have also succeeded in establishing and characterizing hESCs lines. ThehESCs proliferate quickly after being established, so efficient cryopreservationtechnologies are crucial. We use three methods including slow freezing-rapid thawingmethod, vitrification and programmable freezing protocols to cryopreserve hESCs. Then the survial rate of the frozen clumps, the levels of the proliferation anddifferentiation of hESCs after freezing and thawing were compared among these threeprotocols. The pluripotency of hESCs was also identified.There are three parts in this study which includes the establishment of hESCswith the discarded embryos after IVF-ET, the characterization of hESCs and theresearch of comparison of different cryopreservation methods of hESCs.We collected 177 D₃ discarded embryos, in which 47 blastocysts were formed;we thawed 23 frozen embryos, and in which 7 blastocysts were formed . Theestablishment of hESCs line is closely related with the quality of blastocyst and theoriginal growth of ICM. Thus the hESCs derived from good-quality blastocysts couldbe passaged further than that from bad-quality, and the ICM growing faster could bepassaged more efficiently. Using the ICM of good-quality blastocysts, we hadestablished four hESCs lines which named hESCs-060909, hESCs-060927,hESCs-061005 and hESCs-061124 respectively, the hESCs-061124 is derived fromthawed embryo. These four hESCs lines have been propagated to P₂₄, P₁₉, P₁₇ and P₆respectively.The characterization of hESCs lines includes the normal morphology of ES cells,the demonstration of the presence of specific cell surface markers (alkalinephosphatase, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81) and the retainmentof normal karyotype. The hESCs-060909 P₂₀, hESCs-060927 P₁₅ and hESCs-061124P₆ are positive for alkaline phosphatase expression. The hESCs-060909 P₂₀ andhESCs-060927 P₁₅ are negative for SSEA-1, partially positive for SSEA-3 andpositive for SSEA-4 and TRA-1-60. The karyotype of hESCs-060909 P₂₀ is normal(46,XY), the karyotype of hESCs-060927 P₁₅ is normal (46,XX).We use three different methods to evaluate the efficiency of cryopreservation ofhESCs. The 93 hESCs clumps from identical cell line were randomly and averagely allocated to be cryopreserved by slow freezing-rapid thawing method, vitrification orprogrammable freezing protocol. There were 31 hESCs clumps in each group. Wethawed the hESCs clumps after a week, then we compared the survival rate (thesurvival rate=the reclaimed hESCs clumps/the frozen hESCs clumps) of these threegroups: 35.5% hESCs clumps were recovered after vitrification, 16.1% hESCsclumps were recovered after programmable freezing protocol and 12.9% stem cellclumps were recovered after slow freezing-rapid thawing method. The differencebetween vitrification and slow freezing-rapid thawing method was statisticallysignification (P＜0.05), thus the vitrification is an effective method to cryopreservehESCs. We also compared the normal-morphology , the proliferation anddifferentiation level of the hESCs of these three methods. The hESCs colonies aftervitrification not only manifested normal morphology but alao faster growth and lowerlevel of differentiation compared with those colonies after programmable freezingprotocol and slow freezing-rapid thawing method. Finally, we identify thepluripotency of hESCs after thawing including normal karyotype, expression ofAKP, SSEA-3 and SSEA-4.