Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Activation of the unfolded protein response in human acute myeloid leukemia

There is accumulating evidence for the involvement of the unfolded protein response (UPR) in the pathogenesis of many tumor types in humans. This is particularly the case in rapidly growing solid tumors in which the demand for oxygen and nutrients can exceed the supply until new tumor-initiated blood vessels are formed. In contrast, the role of the UPR during leukemogenesis remains largely unknown. Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by the accumulation of somatic mutations in hematopoietic progenitor cells that alter the physiological regulation of self-renewal, survival, proliferation, or differentiation. The CCAAT/enhancer-binding protein alpha (CEBPA) gene is a key myeloid transcription factor and a frequent target for disruption in AML. In particular, translation of CEBPA mRNA can be specifically blocked by binding of the chaperone calreticulin (CALR), a well-established effector of the UPR, to a stem loop structure within the 5' region of the CEBPA mRNA. The relevance of this mechanism was first elucidated in certain AML subtypes carrying the gene rearrangements t(3;21) or inv(16). In our recent work, we could demonstrate the induction of key effectors of the UPR in leukemic cells of AML patients comprising all subtypes (according to the French-American-British (FAB) classification for human AML). The formation of the spliced variant of the X-box binding protein (XBP1s) was detectable in 17.4% (17 of 105) of AML patients. Consistent with an activated UPR, this group had significantly increased expression of the UPR target genes CALR, the 78 kDa glucose-regulated protein (GRP78), and the CCAAT/enhancer-binding protein homologous protein (CHOP). Consistently, in vitro studies confirmed that calreticulin expression was upregulated via activation of the ATF6 pathway in myeloid leukemic cells. As a consequence, CEBPA protein expression was inhibited in vitro as well as in leukemic cells from patients with activated UPR. We therefore propose a model of the UPR being involved in leukemogenesis through induction of calreticulin along the ATF6 pathway, thereby ultimately suppressing CEBPA translation and contributing to the block in myeloid differentiation and cell-cycle deregulation which represent key features of the leukemic phenotype. From a more clinical point of view, the presence of activated UPR in AML patient samples was found to be associated with a favorable disease course.

Survivin in skin pathologies

Survivin is a member of the inhibitor of apoptosis (IAP) protein family acting at the intersection between proliferation and cell survival. This protein exhibits low or undetectable expression in most adult tissues but is increased in the majority of cancers. Suggested to be one of the most cancer-specific proteins identified to date, survivin acts as a signalling node in tumour maintenance and, after first promising results, is now attracting increasing attention as a target in anti-cancer therapy. In the skin, survivin has been implicated in a number of pathological conditions such as psoriasis and tumours of melanocytic and epithelial origin. Its expression can correlate with tumour severity, metastasis and decreased patient survival and has been inversely correlated with the sensitivity to cytotoxic agents used in anti-cancer therapy. Survivin may also be of importance for normal epidermal homeostasis possibly supporting self-renewal of epidermal stem cells. In this review, the authors summarize and discuss current data of survivin in skin biology and provide a comprehensive compilation of survivin expression in skin pathologies with focus on future therapeutical use.

C/EBPalpha and the pathophysiology of acute myeloid leukemia

PURPOSE OF REVIEW: The transcription factor C/EBPalpha controls differentiation and proliferation in normal granulopoiesis in a stage-specific manner. Loss of C/EBPalpha function in myeloid cells in vitro and in vivo leads to a block to myeloid differentiation similar to that which is observed in malignant cells from patients with acute myeloid leukemia. The finding of C/EBPalpha alterations in subgroups of acute myeloid leukemia patients suggests a direct link between critically decreased C/EBPalpha function and the development of the disorder. RECENT FINDINGS: Conditional mouse models provide direct evidence that loss of C/EBPalpha function leads to the accumulation of myeloid blasts in the bone marrow. Targeted disruption of the wild type C/EBPalpha protein, while conserving the dominant-negative 30 kDa isoform of C/EBPalpha, induces an AML-like disease in mice. In hematopoietic stem cells C/EBPalpha serves to limit cell self-renewal. Finally, C/EBPalpha function is disrupted at different levels in specific subgroups of acute myeloid leukemia patients. SUMMARY: There is evidence that impaired C/EBPalpha function contributes directly to the development of acute myeloid leukemia. Normal myeloid development and acute myeloid leukemia are now thought to reflect opposite sides of the same hematopoietic coin. Restoring C/EBPalpha function represents a promising target for novel therapeutic strategies in acute myeloid leukemia.

In utero haematopoietic stem cell transplantation. Experiences in mice, sheep and humans

Early prenatal diagnosis and in utero therapy of certain fetal diseases have the potential to reduce fetal morbidity and mortality. The intrauterine transplantation of stem cells provides in some instances a therapeutic option before definitive organ failure occurs. Clinical experiences show that certain diseases, such as immune deficiencies or inborn errors of metabolism, can be successfully treated using stem cells derived from bone marrow. However, a remaining problem is the low level of engraftment that can be achieved. Efforts are made in animal models to optimise the graft and study the recipient's microenvironment to increase long-term engraftment levels. Our experiments in mice show similar early homing of allogeneic and xenogeneic stem cells and reasonable early engraftment of allogeneic murine fetal liver cells (17.1% donor cells in peripheral blood 4 weeks after transplantation), whereas xenogeneic HSC are rapidly diminished due to missing self-renewal and low differentiation capacities in the host's microenvironment. Allogeneic murine fetal liver cells have very good long-term engraftment (49.9% donor cells in peripheral blood 16 weeks after transplantation). Compared to the rodents, the sheep model has the advantage of body size and gestation comparable to the human fetus. Here, ultrasound-guided injection techniques significantly decreased fetal loss rates. In contrast to the murine in utero model, the repopulation capacities of allogeneic ovine fetal liver cells are lower (0.112% donor cells in peripheral blood 3 weeks after transplantation). The effect of MHC on engraftment levels seems to be marginal, since no differences could be observed between autologous and allogeneic transplantation (0.117% donor cells vs 0.112% donor cells in peripheral blood 1 to 2 weeks after transplantation). Further research is needed to study optimal timing and graft composition as well as immunological aspects of in utero transplantation.

Phenotypic and functional analysis of human fetal liver hematopoietic stem cells in culture

Steady-state hematopoiesis and hematopoietic transplantation rely on the unique potential of stem cells to undergo both self-renewal and multilineage differentiation. Fetal liver (FL) represents a promising alternative source of hematopoietic stem cells (HSCs), but limited by the total cell number obtained in a typical harvest. We reported that human FL nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) could be expanded under simple stroma-free culture conditions. Here, we sought to further characterize FL HSC/SRCs phenotypically and functionally before and following culture. Unexpanded or cultured FL cell suspensions were separated into various subpopulations. These were tested for long-term culture potential and for in vivo repopulating function following transplantation into NOD/SCID mice. We found that upon culture of human FL cells, a tight association between classical stem cell phenotypes, such as CD34(+) /CD38(-) and/or side population, and NOD/SCID repopulating function was lost, as observed with other sources. Although SRC activity before and following culture consistently correlated with the presence of a CD34(+) cell population, we provide evidence that, contrary to umbilical cord blood and adult sources, stem cells present in both CD34(+) and CD34(-) FL populations can sustain long-term hematopoietic cultures. Furthermore, upon additional culture, CD34-depleted cell suspensions, devoid of SRCs, regenerated a population of CD34(+) cells possessing SRC function. Our studies suggest that compared to neonatal and adult sources, the phenotypical characteristics of putative human FL HSCs may be less strictly defined, and reinforce the accumulated evidence that human FL represents a unique, valuable alternative and highly proliferative source of HSCs for clinical applications.

Longitudinal data on telomere length in leukocytes from newborn baboons support a marked drop in stem cell turnover around 1 year of age

Stem cells of various tissues are typically defined as multipotent cells with 'self-renewal' properties. Despite the increasing interest in stem cells, surprisingly little is known about the number of times stem cells can or do divide over a lifetime. Based on telomere-length measurements of hematopoietic cells, we previously proposed that the self-renewal capacity of hematopoietic stem cells is limited by progressive telomere attrition and that such cells divide very rapidly during the first year of life. Recent studies of patients with aplastic anemia resulting from inherited mutations in telomerase genes support the notion that the replicative potential of hematopoietic stem cells is directly related to telomere length, which is indirectly related to telomerase levels. To revisit conclusions about stem cell turnover based on cross-sectional studies of telomere length, we performed a longitudinal study of telomere length in leukocytes from newborn baboons. All four individual animals studied showed a rapid decline in telomere length (approximately 2-3 kb) in granulocytes and lymphocytes in the first year after birth. After 50-70 weeks the telomere length appeared to stabilize in all cell types. These observations suggest that hematopoietic stem cells, after an initial phase of rapid expansion, switch at around 1 year of age to a different functional mode characterized by a markedly decreased turnover rate.

LIF promotes neurogenesis and maintains neural precursors in cell populations derived from spiral ganglion stem cells

BACKGROUND: Stem cells with the ability to form clonal floating colonies (spheres) were recently isolated from the neonatal murine spiral ganglion. To further examine the features of inner ear-derived neural stem cells and their derivatives, we investigated the effects of leukemia inhibitory factor (LIF), a neurokine that has been shown to promote self-renewal of other neural stem cells and to affect neural and glial cell differentiation. RESULTS: LIF-treatment led to a dose-dependent increase of the number of neurons and glial cells in cultures of sphere-derived cells. Based on the detection of developmental and progenitor cell markers that are maintained in LIF-treated cultures and the increase of cycling nestin-positive progenitors, we propose that LIF maintains a pool of neural progenitor cells. We further provide evidence that LIF increases the number of nestin-positive progenitor cells directly in a cell cycle-independent fashion, which we interpret as an acceleration of neurogenesis in sphere-derived progenitors. This effect is further enhanced by an anti-apoptotic action of LIF. Finally, LIF and the neurotrophins BDNF and NT3 additively promote survival of stem cell-derived neurons. CONCLUSION: Our results implicate LIF as a powerful tool to control neural differentiation and maintenance of stem cell-derived murine spiral ganglion neuron precursors. This finding could be relevant in cell replacement studies with animal models featuring spiral ganglion neuron degeneration. The additive effect of the combination of LIF and BDNF/NT3 on stem cell-derived neuronal survival is similar to their effect on primary spiral ganglion neurons, which puts forward spiral ganglion-derived neurospheres as an in vitro model system to study aspects of auditory neuron development.

Robust postmortem survival of murine vestibular and cochlear stem cells

Potential treatment strategies of neurodegenerative and other diseases with stem cells derived from nonembryonic tissues are much less subjected to ethical criticism than embryonic stem cell-based approaches. Here we report the isolation of inner ear stem cells, which may be useful in cell replacement therapies for hearing loss, after protracted postmortem intervals. We found that neonatal murine inner ear tissues, including vestibular and cochlear sensory epithelia, display remarkably robust cellular survival, even 10 days postmortem. Similarly, isolation of sphere-forming stem cells was possible up to 10 days postmortem. We detected no difference in the proliferation and differentiation potential between stem cells isolated directly after death and up to 5 days postmortem. At longer postmortem intervals, we observed that the potency of sphere-derived cells to spontaneously differentiate into mature cell types diminishes prior to the cells losing their potential for self-renewal. Three-week-old mice also displayed sphere-forming stem cells in all inner ear tissues investigated up to 5 days postmortem. In summary, our results demonstrate that postmortem murine inner ear tissue is suited for isolation of stem cells.

Enhancement of titanium implants via bioengineered periodontal tissues

Osseointegration of titanium dental implants into the jaw bone, which is required for maintenance of the implant in the jaw, results in ankylosis. Dental implants are therefore very unlike natural teeth, which exhibit significant movement in response to mechanical forces. The ability to generate periodontal ligament (PDL) tissues onto dental implants would better mimic the functional characteristics of natural teeth, and would likely improve implant duration and function. OBJECTIVES: The objective of this study was to investigate the feasibility of bioengineering PDL tissues onto titanium implant surfaces. METHODS: Bilateral maxillary first and second molars of 8-week old rats were extracted and used to generate single cell suspensions of PDL tissues, which were expanded in culture. Immunohistochemistry and RT-PCR were used to identify putative PDL progenitor/stem cell populations and characterize stem cell properties, including self-renewal, multipotency and stem cell maker expression. Cultured rPDL cells were harvested at third passage, seeded onto Matrigel-coated titanium implants (1.75 mm x 1 mm), and placed into healed M1/M2 extraction sites. Non-cell seeded Matrigel-coated titanium implants served as negative controls. Implants were harvested after 8, 12, or 18 weeks. RESULTS: Cultured rPDL cells expressed the mesenchymal stem-cell marker STRO-1. Under defined culture conditions, PDL cells differentiated into adipogenic, neurogenic and osteogenic lineages. While control implants were largely surrounded by alveolar bone, experimental samples exhibited fibrous PDL-like tissues, and perhaps cementum, on the surface of experimental implants. CONCLUSIONS: PDL contains stem cells that can generate cementum/PDL-like tissue in vivo. Transplantation of these cells might hold promise as a therapeutic approach for the bioengineering of PDL tissues onto titanium implant. Further refinement of this method will likely result in improved dental implant strategies for use of autologous PDL tissue regeneration in humans. This research was supported by CIMIT, and NIH/NIDCR grant DE016132 (PCY), and TEACRS (YL).

Cardiac c-kit+AT2+ cell population is increased in response to ischemic injury and supports cardiomyocyte performance

The expression pattern of angiotensin AT2 receptors with predominance during fetal life and upregulation under pathological conditions during tissue injury/repair process suggests that AT2 receptors may exert an important action in injury/repair adaptive mechanisms. Less is known about AT2 receptors in acute ischemia-induced cardiac injury. We aimed here to elucidate the role of AT2 receptors after acute myocardial infarction. Double immunofluorescence staining showed that cardiac AT2 receptors were mainly detected in clusters of small c-kit+ cells accumulating in peri-infarct zone and c-kit+AT2+ cells increased in response to acute cardiac injury. Further, we isolated cardiac c-kit+AT2+ cell population by modified magnetic activated cell sorting and fluorescence activated cell sorting. These cardiac c-kit+AT2+ cells, represented approximately 0.19% of total cardiac cells in infarcted heart, were characterized by upregulated transcription factors implicated in cardiogenic differentiation (Gata-4, Notch-2, Nkx-2.5) and genes required for self-renewal (Tbx-3, c-Myc, Akt). When adult cardiomyocytes and cardiac c-kit+AT2+ cells isolated from infarcted rat hearts were cocultured, AT2 receptor stimulation in vitro inhibited apoptosis of these cocultured cardiomyocytes. Moreover, in vivo AT2 receptor stimulation led to an increased c-kit+AT2+ cell population in the infarcted myocardium and reduced apoptosis of cardiomyocytes in rats with acute myocardial infarction. These data suggest that cardiac c-kit+AT2+ cell population exists and increases after acute ischemic injury. AT2 receptor activation supports performance of cardiomyocytes, thus contributing to cardioprotection via cardiac c-kit+AT2+ cell population.

Regulation of hematopoietic and leukemic stem cells by the immune system.

Hematopoietic stem cells (HSCs) are rare, multipotent cells that generate via progenitor and precursor cells of all blood lineages. Similar to normal hematopoiesis, leukemia is also hierarchically organized and a subpopulation of leukemic cells, the leukemic stem cells (LSCs), is responsible for disease initiation and maintenance and gives rise to more differentiated malignant cells. Although genetically abnormal, LSCs share many characteristics with normal HSCs, including quiescence, multipotency and self-renewal. Normal HSCs reside in a specialized microenvironment in the bone marrow (BM), the so-called HSC niche that crucially regulates HSC survival and function. Many cell types including osteoblastic, perivascular, endothelial and mesenchymal cells contribute to the HSC niche. In addition, the BM functions as primary and secondary lymphoid organ and hosts various mature immune cell types, including T and B cells, dendritic cells and macrophages that contribute to the HSC niche. Signals derived from the HSC niche are necessary to regulate demand-adapted responses of HSCs and progenitor cells after BM stress or during infection. LSCs occupy similar niches and depend on signals from the BM microenvironment. However, in addition to the cell types that constitute the HSC niche during homeostasis, in leukemia the BM is infiltrated by activated leukemia-specific immune cells. Leukemic cells express different antigens that are able to activate CD4(+) and CD8(+) T cells. It is well documented that activated T cells can contribute to the control of leukemic cells and it was hoped that these cells may be able to target and eliminate the therapy-resistant LSCs. However, the actual interaction of leukemia-specific T cells with LSCs remains ill-defined. Paradoxically, many immune mechanisms that evolved to activate emergency hematopoiesis during infection may actually contribute to the expansion and differentiation of LSCs, promoting leukemia progression. In this review, we summarize mechanisms by which the immune system regulates HSCs and LSCs.