Abstract PO-094: Human pluripotent stem cells acquire malignancy under tumor microenvironment
Authors: Afify Said M. , Hassan Ghmkin , Seno Akimasa , Iwasaki Yoshiaki , Seno Masaharu 
Published: December 2020

Abstract
Cancer stem cell (CSC) theory suggests that cancer tissue contains a subpopulation of stem-like cells with self-renewal and differentiation potential as normal stem cells maintain organs by self-renewing and differentiation. Even in both cases of malignancy and normal the stemness is affected by the niche surrounding the stem cells. In the initial stage no stem cells should be related with cancer. The most important question is “How come the stem cells acquire malignancy?” Here in this study, using human induced pluripotent stem cells (iPSCs) we tried to generate cancer stem cells in the presence of the conditioned medium (CM) derived from human liver cancer cell line Hep 3B cells and human breast cancer cell line BT459 cells. The CM from both cells was collected when the cells became confluent and sterilized by 0.22 micrometer filter. The colonies of human iPSCs were detached from a mitomycin treated fibroblast feeder layer by collagenase and manually dissected into small pieces which were transferred onto ultra-low-attachment dishes. After 7 days the formed embryoid bodies (EBs) of human iPSCs were plated on Matrigel coated plates and maintained in Repro FF2 medium containing 5 ng/ml of basic FGF. Just after 24 hours from plating on Matrigel, the EBs of human iPSCs were treated with CM mixed with Repro FF2, in the ratio of 1:1. The medium was changed every two days with CM for 4 weeks. EBs cultured in the complete Repro FF2 medium without CM were prepared as the control but did not survive for 4 weeks. The survived cells were analyzed by RT-qPCR analysis and Immunofluorescence (IF) staining to confirm the expression of stemness and CSC markers and evaluated the conversion of iPSCs into CSCs. Distinct expression of self-renewal and CSC markers, such as Nanog, SOX2, CD44 and EPCAM, were observed in the survived cells. Sphere formation assay further confirmed the potential of self-renewal in the survived cells. The survived cells possessed differentiation potential into vascular endothelial-like cells. Thus, we concluded the iPSCs were converted into CSCs (hiPS-CSCs). Through these experiments, 2 different conditions derived from 2 independent cell lines Hep3B and BT549 cells were successful to hiPS-CSCs as hiPS-Hep3Bcm and hiPS-BT549cm cells. Furthermore, being treated with paclitaxel, the hiPS-CSCs exhibited potential of chemo-resistance maintaining the intact clonogenic morphology when compared to hiPSC. This study tries to summarize human cancer disease by utilizing the tumor microenvironment in the form of conditioned medium without any genetic manipulation. This should pave the way for the establishment of effective therapeutic agents.
Citation Format: Said M. Afify, Ghmkin Hassan, Akimasa Seno, Yoshiaki Iwasaki, Masaharu Seno. Human pluripotent stem cells acquire malignancy under tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-094.