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Construction and Identification a Culture Method of Non-adherent Microspheres for Human T-cell Lymphoma Stem Cells
Wang Shanshan1, James Yin2, Zhang Hongjie3, Tan Yan1, Hua Qian1*
1Preclinical Medicine College, Beijing University of Traditional Chinese Medicine, Beijing 100029, China; 2General Hospital of Beijing Military Region, Beijing 100700, China; 3Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Abstract: In this work， we established a serum-free culture method for non-adherent microspheres of human T-cell lymphoma (Hut-102) stem cells， and identified them. Enrichment of sphere cells with stem-like properties from human T-cell lymphoma lines were grown in a defined serum-free medium (SFM) with two differentiation inhibitor， CHIR99021 and PD173074 (2i). The expressions of Sox2， Oct4， Nanog， Klf4， Bmi1， C-Myc were measured by RT-PCR. The antigen effect of CD34， CD44 was analyzed by flow cytometry. The cell cycle of Hut-102 cancer stem cells was assessed by PI and nude mouse xenograft assay. Hut-102 sphere enriched in SFM with 2i expressed cancer stem cells markers， including Sox2， Oct4， Nanog， Klf4， Bmi1， C-Myc， the over-expression of Oct4 protein， and high expressions of CD34， CD44 (74.20%， 90.82%). By observing the cell cycle， we found that most cells in the enriched spheres were in G1/G0 phase (64%)， and less cells in S phase (30.56%) compared to the Hut-102 cells. In the T-cell lymphoma derived from caudal vein injection of Hut-102 spheres in athymic nude mice， 67.74%±5.32% of cells expressed Hut-102 specific surface marker (CD3). We reported for the enrichment and characterization of sphere-propagating cells with stem-like properties from Hut-102 cell lines in non-adherent sphere culture with two differentiation inhibitor such as CHIR99021 and PD173074.
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