Establishment Of Human Embryonic Stem Cell Lines And The Relationship Between Mitochondria And Cytoskeleton Dynamic Distribution Pattern During Embryonic Stem Cell Differentiation

ObjectivesTo establish the normal hESCs and disease stem cell model use extra normal fertilization and discarded abnormal fertilizaton embryos, the affected embryos after PGD were also used in this study.To investigation the formation and distribution of cytoskeleton and mitochondria in early embryo, spontaneous differentiation of stem cells and induced differentiation of stem cells, and explore the mechanism of cytoskeleton and mitochondria role during stem cell differentiation.Matetials and methodsEstablishment of hESCs lines: Human embryos were obtained from the Assited Reproduction Center, Affiliated Women Hospital, Zhejiang University, after patients signed Agreement Form. Inner cell mass were isolated from expanded blastocyst which underwent 5 to7 days development after fertilization. Mechanical isolation, immunosurgery isolation and intact embryo culture methods were used to derive the inner cell mass (ICM). ICM outgrowth were separated and passaged gradually. The established lines were characterized when a steady growth of colonies were reached.Identification of hESCs: The following methods were used, alkaline phosphatase activity staining, stem cell markers (SSEA-3,SSEA-4,TRA-1-60,TRA-1-81,Oct-4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex1) detection, karyotype examination,and three germ layers differentiation.Investigation the dynamic distribution of mitochondria and cytoskeleton during embryo development and embryonic stem cell differentiation: Oocytes and different stage of embryo were collected by superovulation and in vitro culture. hESCs and mES were cultured by regular methods, hESCs EB fomation and mES RA induction were used to examine the dynamic distribution of mitochondria and cytoskeleton during stem cell differentiation. We use Mitotrack Red to stain mitochondria, and FITC conjugated anti-β-tubulin Mouse IgM to observe tubulin and microtubule by regular immunofluorescence method. To observe stem cell's ultrastructure, we use normal transmission electron microscopy methods. Results1.Two hESCs isolated from normal embryos, named ZJ01 and ZJ02 respectively, four hESCs lines isolated from abnormal fertilization embryos, named ZJ03, ZJ04, ZJ05, ZJ06 respectively are established. The established lines present morphological features considered characteristic of hES cells: grow in flat, tightly packed colonies with sharp refractory borders, cells are small and round, exhibiting a high nucleus-to-cytoplasm ratio with a one or more prominent nucleoli and typical spacing between the cells. ZJ03 line with number 4 triploid chromosome has the potential to be a disease stem cell model; The pluripotency tests showed that the hES cell lines were able to form EBs when grown in suspension (in vitro differentiation) and immuno-histochemistry revealed the capacity of the EB cells to differentiate into reprsentative of all three germ layers: endoderm, mesoderm and ectoderm.2. From zygote to 8-stage, mitochondria distribute in blastomeres evenly, no polarizing distribution were observed during these stages; no obvious cytoskeleton were seen during these stages except the spindle at mitosis. Cytoskeleton structure forms and was obvious seen from morula to blastocyst, and mitochondria distribution pattern changed from evenly to polarizing distribution.3. Tubulin distributes evenly in undifferentiated stem cells. After stem cells begin to differentiate, expression of tubulin increased, and microtubule appear after further differentiation. Mitochondria distribute evenly in undifferentiated stem cells. After cells begin to differentiate, mitochondria begin to distribute at one pole of the cells, i.e. polarization distribution.In embryoid body (EB), microtubule structure is obvious, mitochondria in EB surround microtubule.After RA induction, different cell types show different microtubule and mitochondria distribution pattern. Common characteristics of RA induced cells are the formation of microtubule and orientated mitochondria distribution.Conclusion1. successfully establish two normal hESCs and four disease model hESCs.2. The expression of tubulin and formation of microtubule are necessary for blastomeres or stem cells differentiation. The number and distribution pattern of mitochondria is the beginning of stem cell differentiation. The direction of stem cell differentiation can be determined by the distribution of cytoskeleton and mitochondria in spontaneous differentiation and induced differentiation stem cells.