Comparative Evaluation Of Human Embryonic Stem Cell Lines And Function Of ID-1 Gene During The Differentiation Of Human Embryonic Stem Cells To Endothelial Cells

PARTⅠ. Comparative Evaluation of Human Embryonic Stem Cell Lines Derived from Zygotes with Normal and Abnormal PronucleiBACKGROUND AND OBJECTIVE:hES cell lines have been derived from normally or abnormally fertilized zygotes. However, the similar and different properties of these two types of hES cell lines are not well-known. It has been reported that a diploid human embryonic stem cell line was derived from a mononuclear zygote. However, it remains uncertain whether morphologically multi-pronuclei fertilized zygotes can be used for generation of normal hES cell lines. To address these questions, we generated 9 hES cell lines from zygotes containing normal (2PN) and abnormal (OPN,1PN,3PN) pronuclei morphologically. A side-by-side comparison would help us to understand these bewilderments.METHODS:Derivation and maintenance of hES cell lines from fertilized zygotes with morphologically normal and abnormal pronuclei. Detect alkaline phosphatase (AP) activity and confirm the expression of several undifferentiated (sternness) markers by immunocytochemistry. Test the multilineage differentiation potential in vivo and in vitro by teratomas formation experiment and RT-PCR analysis of differentiated gene expression in EBs. Karyotypic analysis in order to detect the genetic stability, and short tandem repeat (STR) analysis to show distinct identity of each hES cell lines. Compare cell cycle distribution and proliferative potential by flow cytometry and BrdU incorporation assay. Assess undifferentiated and differentiated genes expression by real-time qPCR. Use the neural directed differentiation method to test the neural differentiating potential in vitro culture.RESULTS:We established nine hES cell lines (mSDU-hES 1-9 cell lines) from 46 fresh blastocysts which were clinically discarded, of which 5 were derived from 2PN-zygotes,2 from OPN-zygotes,1 from 1PN-zygotes, and 1 from 3PN-zygotes. Each cell line of different PN zygotes showed similar round compact colony morphology with a defined border towards the feeder layer. Each of the cell lines was successfully cryopreserved and thawed. A period of replicative crisis was not observed for any of the cell lines. Cells of each hES cell lines possessed high levels of AP activity and were positive to transcription factors and glycolipids markers: OCT-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Karyotyping was performed every 6 month and revealed a normal diploid karyotype in each hES cell line. No chromosome aberration was detected. Each of the nine hES cell lines has its own genetic label. SCID-beige mice were killed ten to twelve weeks after being injected with undifferentiated hES cells of each line to confirm the formation of teratomas containing three embryonic germ lineages, which indicated that each cell line has the totipotential in vivo differentiation. The EBs derived from mSDU-hES 1-8 cell lines expressed OCT4 and the tissue-specific genes representing ectoderm, mesoderm and endoderm. However, the mSDU-hES 9 (2PN)-derived EBs expressed genes representing ectoderm and mesoderm, but not endoderm. The cell cycle distributions of these hES cell lines showed a unique pattern of four cell cycle stages G1, S, G2, and M characterized by a large proportion of cells in S phase and a small proportion of cells in G0/G1 and G2/M phase. Compared to mSDU-hES 5 (2PN) cell line, mSDU-hES 2 (OPN) cell line showed higher proportion of cells in S phase and slightly higher composition of proliferating cells while 1PN and 3PN-zygote-derived hES cell lines showed similar composition of proliferating cells with mSDU-hES 5 (2PN) cell line. mSDU-hES 2 (OPN) cell line may have a shorter cell cycle duration than other cell lines tested due to the higher proliferation rate. hES cell lines derived from normal or abnormal pronuclei zygotes expressed all 11 undifferentiated and differentiated markers, but their gene expression levels were variable. For the most undifferentiated genes OCT4, NANOG, LIN41 and SOX2 there was no significant difference among the hES cell lines. However, compared to other hES cell lines, the expression of DPPA5 was significantly lower in 2PN-derived mSDU-hES 5, while the expression of UTF1 was significantly higher in 3PN-derived mSDU-hES 8. A profound variation in relative abundance of gene expression in differentiated markers of the cell lines was observed. After 3 weeks of neural differentiation of hES cells in vitro culture, both NESTIN+ neural progenitors and TUJ1+ neurons were generated from morphologically normally and abnormally fertilized zygote-derived cell lines. 2PN-zygote-derived and OPN-,1PN- and 3PN-zygote-derived hES cell lines showed similar neural differentiating potential.CONCLUSION:1. All 9 hES cell lines derived from zygotes with normal and abnormal pronuclei shared the majority of these characteristics. They all showed a similar expression pattern of "stemness" markers, including transcription factors and glycolipids markers:OCT-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81; the ability to proliferate and differentiate into cell lineages in ectoderm, endoderm, and mesoderm in differentiating embryoid bodies and teratomas. Under directed neural differentiation, all the cell lines differentiated into neural progenitors and neurons.2. Differences did exist in cell cycle and in the relative abundance of gene expression in undifferentiated and differentiated markers, but such differences were also found among normal hES cell lines. This phenomenon may be determined by the special biological property of different cell lines, but have no direct connection with the normal or abnormal pronuclei-derivation.3. Normal hES cell lines can be developed from fertilized zygotes with morphologically abnormal (OPN,1PN,3PN) pronuclei which are usually excluded from clinical use. PartⅡ. Expression and Function of ID-1 Gene during the Differentiation of Human Embryonic Stem Cells to Endothelial Cells Induced by TGF-betalBACKGROUND AND OBJECTIVE:Inhibitor of differentiation/DNA binding-1 (ID-1) is a specific downstream target gene of activin receptor-like kinase-1 (ALK1) in endothelial cells (ECs) and it can mediate the transforming growth factor-β(TGF-P)/ALK1- induced (and Smad-dependent) migration. However, it remains unclear that how ID-1 plays a part during the differentiation of human embryonic stem cells (hESCs) into ECs induced by TGF-β1. In this study, we used the hESCs differentiation model that recapitulates the developmental steps of vasculogenesis in the early stage of embryo development to explore the role that ID-1 gene plays in the process of TGF-β1 induced hESCs differentiation towards endothelial lineage.METHODS:hESCs culture and establishment of TGF-β1 induced differentiation model of hESCs differentiating into the endothelial lineage. Then analyze the effect of TGF-β1 of different concentrations on the differentiation and vasculogenesis by immunostaining of EBs (embryoid bodies) and differentiated cells. Compare the expression of ID-1 gene and endothelial cell marker, PECAM (platelet/endothelial cell adhesion molecule-1),KDR (kinase insert domain receptor) gene in TGF-β1-induced group and control group by qRT-PCR, and then analyze the effect of TGF-β1 on ID-1 gene. Analyze the kinetics of ID-1 expression during differentiation and vasculogenesis induced by TGF-β1 using qRT-PCR technology. Study the function of ID-1 gene during the differentiation of human embryonic stem cells into ECs induced by TGF-β1 by small interfering RNAs and qRT-PCR technology. Analyze the kinetics of TGF-β1 receptors and some signal proteins expression during differentiation and vasculogenesis stages using qRT-PCR and western blot technology.RESULTS:We have demonstrated that at the early stage of differentiation TGF-β1 may induce the in vitro differentiation of hESCs into ECs by inhibiting expression of ID-1, while at the late stage of differentiation TGF-β1 may stimulate the proliferation and migration of ECs via ALK1/Smad1,5/ID-1 pathway. But TGF-β1 has a negative effect on the angiogenic sprout formation during angiogenesis stage. In addition, in vitro differentiation of hESCs into ECs and expression of ID-1 induced by TGF-β1 are not only time-depended, but also depended on the concentration of TGF-β1. Down-regulation of ID-1 by silence of this gene can lead to accelaration of hESCs differentiation into ECs induced by TGF-β1 and inhibition of proliferation and migration of ECs.CONCLUSION:Taken together, the use of TGF-β1 induced hESCs differentiation model permitted us to dissect the function of ID-1 gene during differentiation process towards endothelial lineage and proliferation process of differentiated ECs. Our present data suggest that TGF-β1 may stimulate the endothelial-oriented differentiation through activated ALK5 pathway during the differentiation process, whereas enhance the proliferation of differentiated ECs by activated TGF-β1/ALK1/ID-1, Smad1/Smad5-dependent signal transduction during the proliferation process. This study used the in vitro hESCs differentiation model may help us to understand some mechanisms of vasculogenesis in the early stage of embryo development.