关键词: 人胚胎干细胞, 成年造血, Runx1c, 报告细胞系, CRISPR-Cas9

Abstract: Runt-related transcription factor 1c (Runx1c) is closely related to definitive hematopoiesis and the production of hematopoietic stem cell (HSC). In order to track the dynamical expression of Runx1c during early hematopoietic development, Runx1c-mNeonGreen reporter cell line derived from human embryonic stem cell (hESC) was efficiently constructed by CRISPR-Cas9 technology combined double-cut plasmid vector with the transient transfer of B-cell lymphoma-extra large (BCL-XL) gene. It was identified and verified by PCR and Sanger sequencing, and the results showed that the construction was successful. Subsequently, immunofluorescence technique, real-time fluorescence quantitative PCR technique, flow cytometry, teratoma assay and digital karyotype analysis technique were used to verify that the reporter cell line still had hESC-like pluripotency and no chromosome abnormality or variation occurred. In addition, the expression of Runx1c in human early hematopoietic development was tracked by flow cytometry through hESC in vitro monolayer induced hematopoietic differentiation. The results showed that the reporter cell line could dynamically track the expression of Runx1c in human early hematopoietic development. The research results provided a new idea for elucidating definitive hematopoiesis and the possibility of producing functional blood cells in vitro.

Key words: human embryonic stem cells, definitive hematopoiesis, Runx1c, reporter cell line, CRISPR-Cas9