CSCs are identified either by surface markers and up regulat

CSCs are identified either by surface markers and up-regulation of transcription factors/other genes involved in stem cell pathways or by functional assays to characterize stem like properties like drug efflux, quiescence and self-renewal (Moserle et al., 2010). Studies have reported enrichment of CSCs by growing them in presence of chemotherapeutic drugs which retains drug resistant side population (Zhang et al., 2010). We established drug resistant side population from leukemic cell lines by enrichment in presence of methotrexate and sorted cetrizine with LSC phenotype expressing CD34 and CD123. Various surface markers for leukemic stem cells have been recently identified (Becker and Jordan, 2011, Zhou and Chng, 2014). Although most of the initial studies have used CD34+/CD38− cells for sorting LSCs (Zhou and Chng, 2014), recent studies have identified more specific LSC markers like CD96 for AML (Hosen et al., 2007), CD123 (Jordan et al., 2000, Du et al., 2011, Hwang et al., 2012), CD90 and CD110 for ALL (Yamazaki et al., 2009), CD47 (Majeti et al., 2009), CLL (van Rhenen et al., 2007), CD32 and CD25 (Saito et al., 2010). However, CD123 represents a unique marker for primitive LSCs and CD34/CD123 markers have been used to isolate LSCs (Jordan, 2007). CD123 is also widely expressed in both AML and ALL (Muñoz et al., 2001). LSCs were confirmed by surface marker analysis, up-regulation of transcription factors and genes involved in stem cell pathways and by functional assays. The greatest challenge in targeting cancer stem cells is their ability to evade cell death induced by radio- or chemotherapy. Targeting apoptotic pathway is an attractive strategy especially due to the over expression of DRs reported in CSCs (Signore et al., 2013, He et al., 2014). Recombinant TRAIL has been used in combination with Ara-c and daunorubicin for AML progenitors (Plasilova et al., 2002) and was effective in breast cancer SCs when c-FLIP was inhibited (Piggott et al., 2011). TRAIL gene armed oncolytic viruses effectively targeted lung cancer stem-like cells (Yang et al., 2015). Gene expression and surface marker analysis showed 4–5 fold higher expression of DR5 receptor in SP-cells compared to non-SP cells in this study. Up-regulation of DR5, induced by cisplatin, restored TRAIL induced cell death in glioblastoma derived stem cells (Ding et al., 2011). Higher expression of DR4 resulted in greater sensitivity to TRAIL in chemotherapy resistant colon cancer SP cells (Sussman et al., 2007). Use of DR5 selective TRAIL peptide mimic with increased affinity was shown to induce apoptosis effectively independent of p53 (Pavet et al., 2010). Selective targeting of DR5 has additional advantage of overcoming TRAIL resistance due to binding decoy receptors (Koschny et al., 2007, Buchsbaum et al., 2006). Hence, we used DR5 selective TRAIL peptide to target and induce apoptotic pathway specifically in drug resistant LSCs, which showed up-regulation of DR5 receptor. LSCs also showed higher expression of CD25 and CD123 in both myeloid and lymphoid leukemic cell lines. CD25 is a unique receptor that is over expressed in most of the hematological malignancies (Erber and Mason, 1988). Interestingly, CD25 was also recently reported to be a potential LSC target since the receptor is selectively over-expressed in LSCs not normal stem cells (Saito et al., 2010). Although many surface markers were reported as therapeutic targets for LSCs, this selective feature of CD25 makes it an ideal target as normal stem cells would be spared. We showed the potential of targeting CD25 in our previous study. Here, we show the effective specific targeting of CD25 in LSCs using IL2 fused with TRAIL peptide mimetic.
Conclusion The following are the supplementary data related to this article.
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Introduction Human (h) pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) potentially provide an unlimited cell source for cell replacement therapies owing to their pluripotency and self-renewal capability. hPSC-derived cardiomyocytes (hPSC-CMs) can now be efficiently generated with directed cardiac differentiation protocols developed by various investigators (Yang et al., 2008; Laflamme et al., 2007; Burridge et al., 2011; Weng et al., 2014). However, hPSC-CMs are structurally and functional immature. For instance, the physical size and nucleation status of hPSC-CMs are strikingly different from mature ventricular (V) CMs in the adult human heart (Kong et al., 2010; Hartman et al., 2015). Comparing to adult VCMs, which are larger in size (~130μm in length) and bi-nucleated in up to 57% of the VCM populations (Schmid and Pfitzer, 1985), hPSC-CMs are 10× smaller in size with mono-nucleation in the majority (Lundy et al., 2013). However, the significance of physical size and nucleation status for the biology of hPSC-CMs has never been addressed. We therefore attempted to increase the physical size and nucleation status of hPSC-VCMs via a chemically-induced cell fusion. The electrophysiological (EP) properties, Ca2+ handling, mitochondrial biogenesis of the multi-nucleated hPSC-VCMs fused syncytia (FS) generated were investigated for consequential phenotypic alterations.