| Objective:To assess the quality, pluripotency, and self-renewal of some of the Human embryonic stem cell(hESC)lines available at Institute of Reproductive and Stem cell Engineering, Central South University, and to analyze the expression pattern of some transcription factors critical for megakaryocytic-erythrocytic differentiation such as FLI-1, RUNX-1, GATA-1,FOG-1,and KLF-1.Method:Human embryonic stem cell lines chHES-8, chHES-22, chHES-26, chHES-51, chHES-55, chHES-60, chHES-137, chHES-254, chHES-268, chHES-281, cgHES-282, chHES-283, chHES-284were cultured on mouse embryonic feeder(MEFs)layer for few days and then immunostained for embryonic stem cell markers. hESC were also subjected to telomerase activity and karyotype analysis.To assess in vitro differentiation, hESC were cultured in DFSR medium without bFGF. After three weeks culture, cells were immunostained for characterization of the three germ layer markers.To assess in vivo differentiation, hESCs were injected intramuscularly into the leg of8-week old mice with severe combined immunodeficiency disease (SCID). The mice were sacrificed8weeks post-injection and the animal grafts were processed for histological analyses by haematoxylin and eosin (H&E) staining. For transcription factor and gene expression testing, to exclude contamination of MEFs, hESCs were transferred to matrigel-plated feeder free culture medium mTeSR for3-4days. Reverse transcriptase PCR (RT-PCR) performed after RNA extraction. PCR products were separated onto agarose gel, stained with ethidium bromide, visualized and photographed on a UV transluminator.Results:hESC lines were stained positive for stem cell markers AKP, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81and OCT-4RT-PCR showed high expression level of pluripotency related genes in all cell lines.Karyotype analysis displayed the normal chromosomal karyotype for different cell lines, i-e46, XY and46,XX.After three weeks in vitro culture for differentiation,immunostaining showed three germ layer markers β-tubulin-Ⅲ,SMA and AFP.After8weeks in vivo, teratoma formed revealing three germ layer tissues, neural tube, cartilage, and glandular epithelium.RT-PCR gel-electrophoresis after FOG-1, GATA-1, KLF-1, FLI-1, Runx-1and GAPDH primers, showed different levels of transcription factor expression.Cell lines chHES-281and chHES-284showed high level expression for transcription factors KLF-1and FOG-1.Cell lines chHES-137showed high level expression for FLI-1and FOG-1.Cell line chHES-8strongly expresses only FLI-1.RUNX-1and GATA-1are either not expressed or expressed in small degrees in all hESC lines. Except chHES-281, chHES-284, most of the other cell lines also did not strongly express KLF-1.Conclusion:1. All hESC lines expressed stem cell markers and genes and had the ability of self-renewal and were pluripotent.2.A11hESC lines were not equivalent and expressed different levels of transcription factors specific for megakaryocytic or erythrocytic lineages.3. As few of the cell lines showed high level expression for the transcription factors specific for either megakaryocytic or erythrocytic pathways, in theory,these particular hESC line should be used for further differentiation to megakaryocytes or erythrocytes respectively.4. As most of the hESC lines did not expressed RUNX-1,GATA-1, KLF-1at high levels.this may be one of the reason of hESC not committing to either megakaryocytic or erythrocytic pathway and are retaining self-renewing ability.5. Our data will help in future studies for understanding the megakaryocytic and erythrocytic differentiation pathways and for the ultimate generation of platelets and RBCs from hESC for clinical use. |
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