Abstract Glioblastoma multiforme, as its name suggests, composes pathologically heterogeneous mixed cells, such as variable degree of cellular and nuclear polymorphism, or so called “gemistocytes” or “giant cells”. Cancer cell regulates dynamic cell-cell interactions, thus, cell-cell dynamic heterogeneity is fundamental questions to know which cells are origin, therapeutic targets or source of recurrences for these decades. Therefore, functional analysis of each heterogenous cells and their role in tumor pathogenesis are critical. To investigate the cellular origin of population diversity, we established 4 subclones from a IDH1 wild type GBM patient. These subclones were subsequently propagated and analyzed. All clones expressed stem cell makers (nestin, sox2, musashi) heterogeneously. The morphology, self-renewal and proliferative capacities of the subclones were varying. FACS analysis showed that high CD133 expression clones tended to have high proliferation and invasive capacities. Most of these clones’ cells expressed high level of CD44, an adhesion molecule in cancer stem cells regulating redox status in cancer cells. Surface expression of CD24 and CD56 showed statistically different pattern. However, these 2 did not enhance their morphology, proliferation capacities and tumorigenesis. On the other hand, cDNA analysis with using 58721 probes showed that the level of COL1A1 and IGFBP7 were matched their morphology and tumorigenesis character. In animal models, xenografts from subclones, tumor-progression/invasion and animal survivals were also different. Some clones invaded contra-lateral hemisphere via tract fiber. Our observations suggest that single cell derived subclones from a patient is capable of producing phenotypically heterogeneous self-renewing progenies in an in vitro and in vivo setting. The functional analysis of the heterogeneous GSC clones may provide a better understanding of GSC biology and novel means for testing of new treatment strategies that focus on the eradication of the true GSCs. Citation Format: Akio Soeda, Akira Hara, Takahiro Kunisada, Toru Iwama. Different tumor-initiating cells from a IDH1 wt glioblastoma patient. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3013. doi:10.1158/1538-7445.AM2014-3013