Background—Nuclear reprogramming provides an emerging strategy to produce embryo-independent pluripotent stem cells from somatic tissue. Induced pluripotent stem cells (iPS) demonstrate aptitude for de novo cardiac differentiation, yet their potential for heart disease therapy has not been tested. Methods and Results—In this study, fibroblasts transduced with human stemness factors OCT3/4, SOX2, KLF4, and c-MYC converted into an embryonic stem cell–like phenotype and demonstrated the ability to spontaneously assimilate into preimplantation host morula via diploid aggregation, unique to bona fide pluripotent cells. In utero, iPS-derived chimera executed differentiation programs to construct normal heart parenchyma patterning. Within infarcted hearts in the adult, intramyocardial delivery of iPS yielded progeny that properly engrafted without disrupting cytoarchitecture in immunocompetent recipients. In contrast to parental nonreparative fibroblasts, iPS treatment restored postischemic contractile performance, ventricular wall thickness, and electric stability while achieving in situ regeneration of cardiac, smooth muscle, and endothelial tissue. Conclusions—Fibroblasts reprogrammed by human stemness factors thus acquire the potential to repair acute myocardial infarction, establishing iPS in the treatment of heart disease. (Circulation. 2009;120:408-416.)

Repair of Acute Myocardial Infarction by Human Stemness Factors Induced Pluripotent Stem Cells

The EphA2 Receptor Drives Self-Renewal and Tumorigenicity in Stem-like Tumor-Propagating Cells from Human Glioblastomas

Glioblastoma multiforme (GBM) is a life-threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem-like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator of mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the mitotic kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase-dependent manner. This MELK-dependent activation of FOXM1 results in a subsequent increase in mitotic regulatory genes in GSCs. MELK-driven FOXM1 activation is regulated by the binding and subsequent trans-phosphorylation of FOXM1 by another kinase PLK1. Using mouse neural progenitor cells (NPCs), we found that transgenic expression of FOXM1 enhances, while siRNA-mediated gene silencing diminishes neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases as cells progress from NPCs, to pretumorigenic progenitors and GSCs. The antibiotic Siomycin A disrupts MELK-mediated FOXM1 signaling with a greater sensitivity in GSC compared to neural stem cell. Treatment with the first-line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cells, and addition of Siomycin A to Temozolomide treatment in mice harboring GSC-derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1-dependent manner and thus, this protein complex is a potential therapeutic target for GBM.

/pdf/melk-dependent-foxm1-phosphorylation-is-essential-for-2rg7cuaiyp.pdf

MELK-dependent FOXM1 phosphorylation is essential for proliferation of glioma stem cells.

Glioblastoma multiforme contains a subpopulation of cancer stem-like cells (CSC) believed to underlie tumorigenesis and therapeutic resistance. Recent studies have localized CSCs in this disease adjacent to endothelial cells (EC) in what has been termed a perivascular niche, spurring investigation into the role of EC-CSC interactions in glioblastoma multiforme pathobiology. However, these studies have been limited by a lack of in vitro models of three-dimensional disease that can recapitulate the relevant conditions of the niche. In this study, we engineered a scaffold-based culture system enabling brain endothelial cells to form vascular networks. Using this system, we showed that vascular assembly induces CSC maintenance and growth in vitro and accelerates tumor growth in vivo through paracrine interleukin (IL)-8 signaling. Relative to conventional monolayers, endothelial cells cultured in this three-dimensional system not only secreted enhanced levels of IL-8 but also induced CSCs to upregulate the IL-8 cognate receptors CXCR1 and CXCR2, which collectively enhanced CSC migration, growth, and stemness properties. CXCR2 silencing in CSCs abolished the tumor-promoting effects of endothelial cells in vivo, confirming a critical role for this signaling pathway in GMB pathogenesis. Together, our results reveal synergistic interactions between endothelial cells and CSCs that promote the malignant properties of CSCs in an IL-8-dependent manner. Furthermore, our findings underscore the relevance of tissue-engineered cell culture platforms to fully analyze signaling mechanisms in the tumor microenvironment.

https://cancerres.aacrjournals.org/content/canres/73/23/7079.full.pdf

Glioblastoma Stem Cells Are Regulated by Interleukin-8 Signaling in a Tumoral Perivascular Niche

Radiotherapy for the treatment of cancer is undergoing an evolution, shifting to the use of heavier ion species. For a plethora of malignancies, current radiotherapy using photons or protons yields marginal benefits in local control and survival. One hypothesis is that these malignancies have acquired, or are inherently radioresistant to low LET radiation. In the last decade, carbon ion radiotherapy facilities have slowly been constructed in Europe and Asia, demonstrating favorable results for many of the malignancies that do poorly with conventional radiotherapy. However, from a radiobiological perspective, much of how this modality works in overcoming radioresistance, and extending local control and survival are not yet fully understood. In this review, we will explain from a radiobiological perspective how carbon ion radiotherapy can overcome the classical and recently postulated contributors of radioresistance (α/β ratio, hypoxia, cell proliferation, the tumor microenvironment and metabolism, and cancer stem cells). Furthermore, we will make recommendations on the important factors to consider, such as anatomical location, in the future design and implementation of clinical trials. With the existing data available we believe that the expansion of carbon ion facilities into the United States is warranted.

/pdf/bringing-the-heavy-carbon-ion-therapy-in-the-radiobiological-2rtpjy6hfw.pdf

Bringing the heavy: carbon ion therapy in the radiobiological and clinical context

It has been hypothesized that cancer stem cells (CSC) may account for the pathogenesis underlying various tumors, including GBM. Markers of these CSCs can be potentially used as therapeutic targets. In this review, we discuss the most recent information regarding CSCs, their molecular biology and their potential role in GBM.

Understanding the role of tumor stem cells in glioblastoma multiforme: a review article

Evidence suggests that stem-like cells are responsible for initiation, maintenance and recurrence of solid tumors, including Glioblastoma Multiforme (GBM). GBM is an intractable, highly lethal tumor of the central nervous system. Although epidermal growth factor receptor (EGFR) is highly expressed in many GBMs, anti-EGFR therapies have been unsuccessful as treatment. Few studies have examined EGFR activation in GBM stem cells (GSCs) to determine if patient-specific GSCs are amenable to anti-EGFR therapy pre-clinically. We hypothesized that EGFR activation in GSCs varied between patients and was an important determinant of responsiveness to anti-EGFR therapy. Cell cycle and apoptosis analysis was performed on tumor-spheres by immuncytochemistry in the presence and absence of the AG1478. Second messenger pathways operative in these processes were elucidated by immunoblotting. EGFR activated AKT and inactivated GSK3beta in EGFR+/PTEN+ GSCs. AG1478 and erlotinib significantly decreased the total number of tumor-spheres that EGFR+/ PTEN+ GSCs generated and the rate of sphere formation. Inhibition of EGFR signaling by AG1478 increased GSC senescence and apoptosis, likely via inhibition of AKT and activation of GSK3beta. Sphere formation by EGFR-/ PTEN- GSCs was independent of EGF stimulation, but dependant on B27 growth supplement. Our data suggest that EGFR+/PTEN+ GSCs are susceptible to anti-EGFR therapy in vitro.

EGFR signaling is differentially activated in patient-derived glioblastoma stem cells.

It has been postulated that brain tumor stem cells (TSCs) may be the population of cells responsible for the maintenance and recurrence of glioblastoma multiforme (GBM). The purpose of this study was to optimize a reproducible protocol for generating TSCs for their subsequent transfection or transduction. Patient GBMs were enzymatically and mechanically dissociated and tumor spheres were resuspended in appropriate media and analyzed to ensure they met stem cell criteria. These cells were then transfected with a plasmid or transduced with a viral vector to introduce a previously absent gene and then allowed to form tumor spheres. Tumor spheres were generated from patient GBMs without contamination. These cells met stringent criteria as stem cells, including multipotentiality and self-renewal. High efficiency transfection and transduction of tumor spheres was possible, even at the core of the sphere. This allowed for the introduction of new genes to the TSCs, as evidenced by fluorescent microscopy and Western blot analysis. This study is a guide to optimize the generation of patient derived GBM tumor spheres without RBC and dead cell contamination. GBM TSCs within tumor spheres can easily be transfected with plasmids or transduced with a virus. This is important from a therapeutic perspective if gene replacement is to be successful in replacing genes lost in GBM progression or to knock down or silence genes that are over-expressed in malignant brain tumors.

Robel Beyene

Papers

Understanding the role of tumor stem cells in glioblastoma multiforme: a review article

EGFR signaling is differentially activated in patient-derived glioblastoma stem cells.

Optimization of glioblastoma multiforme stem cell isolation, transfection, and transduction

Disease-specific induced pluripotent stem cells.

Acute pain consult and management is associated with improved mortality in rib fracture patients