Role of nodal signaling and the microenvironment underlying melanoma plasticity

The incidence of melanoma has increased dramatically over the last 50 yr, and although melanoma accounts for only 10% of all skin cancers, it is responsible for over 80% of skin cancer deaths. Recent studies have uncovered critical molecular events underlying melanocytic transformation and melanomagenesis. Among these noteworthy observations are the acquisition of stem cell-associated proteins, such as the Notch receptors and Nodal, which have also been implicated in melanoma progression. For example, we have demonstrated that Nodal expression is limited to invasive vertical growth phase and metastatic melanoma lesions, and that inhibition of Nodal signaling promotes the reversion of metastatic melanoma cells toward a more differentiated, less invasive non-tumorigenic phenotype. In addition, molecular cross-talk exists between the Notch and Nodal signaling pathways. Interestingly, the acquisition of stem cell-associated plasticity is often acquired via epigenetic mechanisms, and is therefore receptive to reprogramming in response to embryonic microenvironments. Here, we review the concept of melanoma plasticity, with an emphasis on the emerging role of Nodal as a regulator of melanoma tumorigenesis and progression, and present findings related to epigenetic reprogramming.     

A blockade in Wnt signaling is activated following the differentiation of F9 teratocarcinoma cells

Aberrant activation of the Wnt signaling pathway is a common event in human tumor progression. Wnt signaling has also been implicated in maintaining a variety of adult and embryonic stem cells by imposing a restraint to differentiation. To understand the effect of Wnt signaling on the differentiation of epithelial cells, we used mouse teratocarcinoma F9 cells as a model. The F9 cells can be differentiated into visceral endoderm (VE) resembling absorptive columnar epithelial cells. We performed comparative gene expression analysis on retinoic acid-differentiated and undifferentiated F9 cells and confirmed that markers of VE and intestinal epithelium were induced upon differentiation. The induction of these markers by retinoic acid was reduced in the presence of Wnt, although Wnt alone did not change their expression. This suggests that Wnt signaling inhibited the differentiation of F9 cells by altering gene expression. This inhibition was also reflected in the morphology of the F9 cells as their apical-basal polarity was disrupted by inclusion of Wnt during differentiation. These results support a model in which Wnt modulates the expression of genes required for normal terminal differentiation of the stem cells. However, it follows that progenitor cells must escape from Wnt signaling to attain the differentiated state. Accordingly, we found that differentiated F9 cells no longer responded to Wnt and that a blockade in Wnt signaling occurred upstream of Axin. Consistent with this, Wnt negative regulators, such as Dickkopf-1 and Disabled-2, were induced upon the differentiation of F9 cells. We propose that a similar system to produce Wnt inhibitors regulates homeostasis of certain stem cell compartments in vivo.     

Fetal adnexa derived stem cells from domestic animal: progress and perspectives

The fetal adnexa such as umbilical cord, amnion and amniotic fluid have been proposed as ideal sources of different stem cell lineages. Use of adnexal tissue has many potential advantages, including the noninvasive nature of the isolation procedure, the large tissue mass from which cells can be harvested with high efficiency and the potential of these cells to differentiate. Moreover, particularly in human medicine, the harvesting of these tissues is more ethically acceptable making these sources of stem cells very attractive for regenerative therapies and biotechnological applications. The adnexal tissue cells preserve some of the characteristics of the primitive embryonic layers from which they originate. Indeed, many studies indicate that these stem cells exhibit some features of embryonic stem cells as expression of embryonic markers and proliferation capability, without showing immunogenicity. However, the differentiation potential of these cells, either in vivo or in vitro, is intermediate between the pluripotent embryonic stem cells and the multipotent adult stem cells. Non-embryonic extra-fetal derived stem cells have opened new perspectives for developmental biology and for regenerative medicine, not only in humans but also in animals. In this update, we report the state of the art of fetal adnexa-derived stem cells from domestic animals and analyze their applications and potential uses in veterinary medicine.     

Stemness,fusion and renewal of hematopoietic and embryonic stem cells

Development of replacement cell therapies awaits the identification of factors that regulate nuclear reprogramming and the mechanisms that control stem cell renewal and differentiation. Once such factors and signals will begin to be elucidated, new technologies will have to be envisaged where uniform differentiation of adult or embryonic stem cells along one differentiation pathway can be induced. Controlled differentiation of stem cells will require the engineering of niches and extracellular signal combinations that would amplify a particular signaling network and allow uniform and selective differentiation. Three recent advances in stem cell research open the possibility to approach engineering studies for cell replacement therapies. Fusion events between stem cells and adult cells or between adult and embryonic stem cells have been shown to result in altered fates and nuclear reprogramming of cell hybrids. Hematopoietic stem cells were shown to require Wnt signaling in order to renew. The purification of Wnt proteins would allow their use as exogenous purified cytokines in attempts to amplify stem cells before bone marrow transplantation. The homeodomain protein Nanog has been shown to be crucial for the embryonic stem cell renewal and pluripotency. However, the cardinal question of how stemness is preserved in the early embryo and adult stem cells remains opened.     

Lineage tracing reveals the dynamic contribution of Hes1+ cells to the developing and adult pancreas

Notch signaling regulates numerous developmental processes, often acting either to promote one cell fate over another or else to inhibit differentiation altogether. In the embryonic pancreas, Notch and its target gene Hes1 are thought to inhibit endocrine and exocrine specification. Although differentiated cells appear to downregulate Hes1, it is unknown whether Hes1 expression marks multipotent progenitors, or else lineage-restricted precursors. Moreover, although rare cells of the adult pancreas express Hes1, it is unknown whether these represent a specialized progenitor-like population. To address these issues, we developed a mouse Hes1(CreERT2) knock-in allele to inducibly mark Hes1(+) cells and their descendants. We find that Hes1 expression in the early embryonic pancreas identifies multipotent, Notch-responsive progenitors, differentiation of which is blocked by activated Notch. In later embryogenesis, Hes1 marks exocrine-restricted progenitors, in which activated Notch promotes ductal differentiation. In the adult pancreas, Hes1 expression persists in rare differentiated cells, particularly terminal duct or centroacinar cells. Although we find that Hes1(+) cells in the resting or injured pancreas do not behave as adult stem cells for insulin-producing beta (β)-cells, Hes1 expression does identify stem cells throughout the small and large intestine. Together, these studies clarify the roles of Notch and Hes1 in the developing and adult pancreas, and open new avenues to study Notch signaling in this and other tissues.     

BMP signaling is required for controlling somatic stem cell self-renewal in the Drosophila ovary

BMP signaling is essential for promoting self-renewal of mouse embryonic stem cells and Drosophila germline stem cells and for repressing stem cell proliferation in the mouse intestine and skin. However, it remains unknown whether BMP signaling can promote self-renewal of adult somatic stem cells. In this study, we show that BMP signaling is necessary and sufficient for promoting self-renewal and proliferation of somatic stem cells (SSCs) in the Drosophila ovary. BMP signaling is required in SSCs to directly control their maintenance and division, but is dispensable for proliferation of their differentiated progeny. Furthermore, BMP signaling is required to control SSC self-renewal, but not survival. Moreover, constitutive BMP signaling prolongs the SSC lifespan. Therefore, our study clearly demonstrates that BMP signaling directly promotes SSC self-renewal and proliferation in the Drosophila ovary. Our work further suggests that BMP signaling could promote self-renewal of adult stem cells in other systems.     

Characterization of calcium signals in human embryonic stem cells and in their differentiated offspring by a stably integrated calcium indicator protein

Intracellular calcium signaling pathways play a major role in cellular responses such as proliferation, differentiation and apoptosis. Human embryonic stem cells (hESC) provide new possibilities to explore the development and differentiation of various cell types of the human body. Intracellular calcium responses to various ligands and the calcium signaling pathways, however, have not been thoroughly studied in embryonic stem cells and in their differentiated progenies. In our previous work we demonstrated that the use of the fluorescent calcium indicator Fluo-4 with confocal microscopy allows sensitive and reliable measurements of calcium modulation in human embryonic stem cells and stem-cell derived cardiomyocytes. Here we developed a human embryonic stem cell line stably expressing a genetically encoded Ca^2 + indicator (GCaMP2) using a transposon-based gene delivery system. We found that the differentiation properties were fully preserved in the GCaMP2-expressing hESC lines and Ca imaging could be performed without the need of toxic dye-loading of the cells. In undifferentiated hES cells the calcium signals induced by various ligands, ATP, LPA, trypsin or angiotensin II were comparable to those in Fluo-4 loaded cells. In accordance with previous findings, no calcium signal was evoked by thrombin, histamine or GABA. Cardiomyocyte colonies differentiated from hES-GCaMP2 cells could be recognized by spontaneous contractions and Ca^2 + oscillations. GCaMP2-expressing neural cells were identified based on their morphological and immuno-staining properties and Ca signals were characterized on those cells. Characteristics of both the spontaneous and ligand-induced Ca^2 + signals, as well as their pharmacological modification could be successfully examined in these model cells by fluorescence imaging.     

Antiproliferative and cytotoxic effects of different type cytostatics on mouse pluripotent stem and teratocarcinoma cells

Pluripotent stem cells are able to proliferate indefinitely and differentiate in vitro into various cell types. However, in most cases in vitro differentiation of the pluripotent stem cells is asynchronous and incomplete, and the residual undifferentiated cells can initiate teratoma development after transplantation into recipients. These features of the pluripotent stem cells are the major issue for development of safe cell therapy technologies based on pluripotent stem cells. Considering significant resemblance of growth rates of pluripotent stem and cancer cells we investigated antiproliferative and cytotoxic effects of different type cytostatics (mitomycin C, etoposide, vinblastine and cycloheximide) on the undifferentiated and differentiating mouse embryonic stem cells, embryonic germ cells, blastocyst and on mouse embryonal teratocarcinoma cells and mouse embryonic fibroblasts. The findings showed that all cytostatics used induced both antiproliferative effects and acute toxic processes in undifferentiated pluripotent stem cells and embryonal teratocarcinoma cells whereas these effects were less in differentiating embryonic stem cells and embryonic fibroblast. Moreover, the trophoblast cells of mouse blastocysts were less sensitive to damaging effects of cytostatics than inner cell mass cells. The examination of deferred effects of cytostatics revealed that the effects of mitomycin C, etoposide and vinblastine, but not cycloheximide, were irreversible because survived cells were not able to proliferate. Nevertheless, the numbers of embryonic fibroblasts exposed to etoposide or vinblastine remained unchanged while vast majority of undifferentiated pluripotent cells treated underwent apoptosis. Thus, diverse effects of etoposide and vinblastine on the undifferentiated pluripotent stem cells and differentiated embryonic cells allow us to consider these cytostatics and their analogs as drug-candidates for selective elimination of the residual undifferentiated pluripotent stem cells from population of differentiating cells. These findings demonstrate for the first time the possibility of selective elimination of undifferentiated pluripotent stem cells using cytostatic drugs approved for clinic practice. However, to improve effectiveness and safety of this approach and to prevent mutagenic, carcinogenic and teratogenic effects on undifferentiated pluripotent stem cells and their differentiated cell derivatives large-scale studies of cytostatic effects using different experimental design and active doses must be performed.     

Stem cells in diabetes: what has been achieved

Beta-cell replacement therapy via islet transplantation has received renewed interest due to the recent improved success. In order to make such a therapy available to more than a few of the thousands of patients with diabetes, new sources of insulin-producing cells must be readily available. The most promising sources are stem cells, with efforts of deriving new beta-cells from both embryonic and adult stem cells. Several groups have reported generating insulin-producing cells from mouse embryonic stem cells. The strategies in the first two acclaimed reports were very different. One strategy, used by Soria's group, is gene trapping in which an introduced antibiotic resistance under the control of the insulin promoter allowed the selection of insulin-expressing cells that had spontaneously differentiated within embryoid bodies. Another strategy, used by McKay's group, manipulated culture conditions in a multistep protocol used for generating neural cells but with changed final conditions. Since these reports, there have been modifications of the protocols in efforts to improve the yields and maturity of the resulting cells. While it is unclear if the insulin-producing cells in any of these studies are truly mature beta-cells, these studies show the clear potential of embryonic stem cells and support optimism that similar results will be possible with human embryonic stem cells. We know that new beta-cells are generated throughout adult life, but the identity of adult pancreatic stem cells has been elusive. The potential for expansion and differentiation of pluripotent adult stem cells, whether from bone marrow or as non-pancreas tissue resident SP cells, is being explored but has not yet yielded insulin-producing tissue. In contrast, insulin-producing cells have been generated in vitro from adult pancreatic tissues. We have been examining the hypothesis that the functional source for new beta-cells in the adult pancreas are mature duct epithelial cells that have regressed or lost their mature phenotype after replication. Others have isolated putative stem cells from islets and ducts. For adult cells the issue of expansion as well as of differentiation is a question. The field of generating new beta-cells from stem cells, either embryonic or adult, is still in its infancy. Each new report has been met with a mixture of excitement and skepticism. With continued efforts and rigorous assessments, hopefully the potential of generating enough new beta-cells from stem cells will be realized.     

Differentiation and isolation of hepatic-like cells from human embryonic stem cells

Human embryonic stem cells are pluripotent cells that can serve as a cell source for transplantation medicine, and as a tool to study human embryogenesis. We investigate here the potential of human embryonic stem cells to differentiate into hepatic cells. We have characterized the expression level of liver-enriched genes in undifferentiated and differentiated human embryonic stem cells by DNA microarrays. Our analysis revealed a subset of fetal hepatic enriched genes that are expressed in human embryonic stem cells upon differentiation into embryoid bodies. In order to isolate the hepatic-like cells, we introduced a reporter gene regulated by a hepatocyte-specific promoter into human embryonic stem cells. We isolated clones of human embryonic stem cells that express enhanced green fluorescent protein upon in vitro differentiation. Through immunostaining, we showed that most of these cells express albumin, while some cells still express the earlier expressed protein alpha-fetoprotein. Using fluorescence activated cell sorter, we were able to sort out the fluorescent differentiated cells and expand them for a few more weeks. This is the first report to demonstrate the possibility of purifying differentiated derivatives of human embryonic stem cells and culturing them further. Through confocal microscopy, we detected clusters of hepatic-like cells in 20-day-old embryoid bodies and in teratomas. As observed during embryonic development, we showed that in teratomas, the hepatic-like endodermal cells develop next to cardiac mesodermal cells. In order to examine the secreted factors involved in the induction of hepatic differentiation, human embryonic stem cells were grown in the presence of various growth factors, demonstrating the potential involvement of acidic fibroblast growth factor in the differentiation. In conclusion, given certain growth conditions and genetic manipulation, we can now differentiate and isolate hepatic-like cells from human embryonic stem cells.     

Umbilical cord blood stem cells: Towards a proteomic approach

The first umbilical cord blood (UCB) transplant to a sibling with Fanconi's anaemia in 1988 represented a breakthrough in the field of transplantation. Thereon, several transplants have been performed with UCB-derived hematopoietic stem cells (HSCs) and a plethora of studies have investigated the plasticity of UCB-derived stem and progenitor cells. However, these studies have not been hitherto translated into clinical trials and, although UCB is routinely used as an alternative source of HSCs, no substantial advances have been made in the field of clinical regenerative medicine. The real deal is the lack of knowledge about the molecular processes governing the events of differentiation which transform immature UCB stem cells into terminally-committed hematopoietic, muscle, bone and nervous cells. In order to fill this void, several studies have been recently focused on the identification of the peculiar proteomic profile of UCB-derived stem cells.Hereby, we concisely review recent proteomic surveys addressing UCB-derived stem and progenitor cells.Notably, comparative studies detected a wider spectrum of proteins in immature cells rather than in more differentiated populations, as if maturation events could represent a bottleneck to protein expression. Future research projects should try to shed light on these processes and their completion could pave the way for unprecedented treatments.     

BMP signaling and stem cell regulation

Stem cells play an essential role in cellular specialization and pattern formation during embryogenesis and in tissue regeneration in adults. This is mainly due to a stem cell's ability to replenish itself (self-renewal) and, at the same time, produce differentiated progeny. Realization of these special stem cell features has changed the prospective of the field. However, regulation of stem cell self-renewal and maintenance of its potentiality require a complicated regulatory network of both extracellular cues and intrinsic programs. Understanding how signaling regulates stem cell behavior will shed light on the molecular mechanisms underlying stem cell self-renewal. In this review, we focus on comparing the progress of recent research regarding the roles of the BMP signaling pathway in different stem cell systems, including embryonic stem cells, germline stem cells, hematopoietic stem cells, and intestinal stem cells. We hope this comparison, together with a brief look at other signaling pathways, will bring a more balanced view of BMP signaling in regulation of stem cell properties, and further point to a general principle that self-renewal of stem cells may require a combination of maintenance of proliferation potential, inhibition of apoptosis, and blocking of differentiation.     

Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome

In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling networks also requires quantitation of these phosphorylation events. In this article, we outline several methods for enrichment of phosphorylated proteins and peptides and discuss various options for their identification and quantitation with special emphasis on mass spectrometry-based techniques.     

Transmitochondrial embryonic stem cells containing pathogenic mtDNA mutations are compromised in neuronal differentiation

Objectives:  Defects of the mitochondrial genome (mtDNA) cause a series of rare, mainly neurological disorders. In addition, they have been implicated in more common forms of movement disorders, dementia and the ageing process. In order to try to model neuronal dysfunction associated with mitochondrial disease, we have attempted to establish a series of trans mitochondrial mouse embryonic stem cells harbouring pathogenic mtDNA mutations.
Materials and methods:  Trans mitochondrial embryonic stem cell cybrids were generated by fusion of cytoplasts carrying a variety of mtDNA mutations, into embryonic stem cells that had been pretreated with rhodamine 6G, to prevent transmission of endogenous mtDNA. Cybrids were differentiated into neurons and assessed for efficiency of differentiation and electrophysiological function.
Results:  Neuronal differentiation could occur, as indicated by expression of neuronal markers. Differentiation was impaired in embryonic stem cells carrying mtDNA mutations that caused severe biochemical deficiency. Electrophysiological tests showed evidence of synaptic activity in differentiated neurons carrying non-pathogenic mtDNA mutations or in those that caused a mild defect of respiratory activity. Again, however, neurons carrying mtDNA mutations that resulted in severe biochemical deficiency had marked reduction in post-synaptic events.
Conclusions:  Differentiated neurons carrying severely pathogenic mtDNA defects can provide a useful model for understanding how such mutations can cause neuronal dysfunction.     

Quantitative phosphoproteomics--an emerging key technology in signal-transduction research

Protein phosphorylation is the most important type of reversible post-translational modification involved in the regulation of cellular signal-transduction processes. In addition to controlling normal cellular physiology on the molecular level, perturbations of phosphorylation-based signaling networks and cascades have been implicated in the onset and progression of various human diseases. Recent advances in mass spectrometry-based proteomics helped to overcome many of the previous limitations in protein phosphorylation analysis. Improved isotope labeling and phosphopeptide enrichment strategies in conjunction with more powerful mass spectrometers and advances in data analysis have been integrated in highly efficient phosphoproteomics workflows, which are capable of monitoring up to several thousands of site-specific phosphorylation events within one large-scale analysis. Combined with ongoing efforts to define kinase-substrate relationships in intact cells, these major achievements have considerable potential to assess phosphorylation-based signaling networks on a system-wide scale. Here, we provide an overview of these exciting developments and their potential to transform signal-transduction research into a technology-driven, high-throughput science.